Giulio Mazzarino was born in Pescina, then part of the Kingdom of Naples, where his parents were travelling, but was raised in Rome. His father Pietro was a notary with connections to the Colonna, who became chamberlain to the Constable Filippo I Colonna and gained an easy situation for his family; Mazarin never forgot that the basis of his fortune in life was the patronage of the Colonna, who had provided his father with a wife, Ortensia Buffalini, of a noble family of Città di Castello in Umbria with an ample dowry. Mazarin studied at the Jesuit College in Rome, though he declined to join their order. At seventeen he accompanied Girolamo Colonna, one of the sons of Filippo I Colonna, to the university of Alcalá de Henares in Spain, to serve as his chamberlain. His stay was brief; a notary who had advanced some cash to cover gaming debts urged the charming and personable young Mazzarino to take his daughter as bride, with a substantial dowry. Later Mazarin frequented the University of Rome La Sapienza, gaining the title of Doctor in jurisprudence but gaining loose habits of serious gambling in the meantime. Mazarin followed Filippo I Colonna as captain of infantry in his regiment during the war in Monferrato of 1628, over the succession to Mantua. During this war he gave proofs of much diplomatic ability, and Pope Urban VIII entrusted him, in 1629, with the difficult task of putting an end to the war of the Mantuan succession.

The Emperor Ferdinand II, the duke of Savoy, Charles Emmanuel I and Ferdinand II of Guastalla, the papal candidate for the duchy, were ranged against Louis XIII in aid of Charles Gonzaga, duc de Nevers, the opposing candidate. Urban VIII sent troops into the Valtelina. At the time, Anna Colonna, daughter of Filippo I Colonna, was married to Urban's nephew, and the Pope now made her brother, Girolamo Colonna, archbishop of Albano and a new cardinal. The Cardinal was sent to Monferrat as papal legate, to treat of peace between France and Spain in the matter of Mantua, and insisted that Mazarin be attached to his legation as secretary. In passing between the armed camps to achieve an accommodation, Mazarin detected the weakness of the Spanish general, the marqués de Santa-Cruz, and perceived that he desired to come to terms without exposing his army to combat. By emphasizing French strengths in the Spanish camp, Mazarin effected the treaty of Cherasco, 6 April 1631, in which the Emperor and the Duke of Savoy recognized the possession of Mantua and part of Monferrat by Charles Gonzaga and the French occupation of the strategic stronghold of Pinerolo, the gate to the valley of the Po, to the great satisfaction of Richelieu and the King of France. Richelieu was in particular impressed by the young man's resourceful ruses, and asked him to come to Paris, where he received him with great demonstrations of affection, promised him great things and gave him a gold chain with the portrait of the King, some jewels and a valuable ceremonial sword.

As papal vice-legate at Avignon (1632), and nuncio extraordinary in France (1634), Mazarin was perceived as an extension of Richelieu's policy. Under Habsburg pressure, Mazarin was sent back to Avignon, where he was dismissed by Urban VIII on January 17, 1636.

After serving in the papal army and diplomatic service and as nuncio at the French court (1634–36), he entered the service of France and made himself valuable to King Louis XIII's chief minister, Cardinal Richelieu, who brought him into the council of state. Richelieu, who felt the weight of his years, though he was as assiduous in the King's service as ever, detected in Mazarin a likely aide in carrying on government. He confided to the young man several sensitive missions, in which Mazarin acquitted himself well, then presented him to the King, who was well pleased with Mazarin. Ever as deft at the gaming table as with diplomacy, one evening his winnings were so great that a crowd gathered to see the stacks of gold écus, attracting the attention of the Queen; in her presence, Mazarin risked all, and won. He attributed his winnings to the Queen's presence, and in thanks, offered her fifty thousand écus. The Queen demurred, Mazarin pressed, and she accepted. Several days later, Mazarin quietly received a great deal more than he had given. Thus he was affirmed in the favour of the King, the court and above all of Anne of Austria, who would soon be regent. Mazarin sent to his father in Rome a great sum of money and a casket of jewels, for which he always had a great fondness, as dowry for his three sisters. Service to the King of France seemed to him the easiest route to a cardinal's hat, his constant ambition. Richelieu, in spite of his fondness and admiration for Mazarin, was loath to crown his career so early; he offered a bishopric worth 30,000 écus a year. Mazarin, who aspired to more, for his part, turned it aside amiably. In 1636 he returned to Rome, with the thought of attaching himself to Cardinal Antonio, nephew of the pope, with an eye to preferment by that route.

In 1640 Richelieu sent him to Savoy, where the regency of Christine, the Duchess of Savoy, and sister of Louis XIII, was disputed by her brothers-in-law, the princes Maurice and Thomas of Savoy, and he succeeded not only in firmly establishing Christine but in winning over the princes to France. This great service was rewarded by his promotion to the rank of cardinal on the presentation of the King of France in December 1641. Soon after, he returned to Rome. His residence in Rome did not last long, as he returned to Paris in December 1642, after the death of Richelieu, succeeding him as Chief Minister of France.

King Louis XIII died in 1643. His successor, Louis XIV, was only five years old at the time and his mother, Anne of Austria, ruled in his place until he came of age. Mazarin helped Anne expand her power from the more limited power her husband had left her. Mazarin functioned essentially as the co-ruler of France alongside the queen during the regency of Anne, and until his death in 1661 at Vincennes, Mazarin effectively directed French policy alongside the monarch. His modest manner contrasted with the imperious Richelieu, and Anne was so fond of him and so intimate in her manner with him, that there were long-standing rumors that they had been secretly married and that the Dauphin was their offspring.

Mazarin continued Richelieu's anti-Habsburg policy and laid the foundation for Louis XIV's expansionist policies. The victories of Condé and Turenne brought the French party to the bargaining table at the conclusion of the Thirty Years' War with the Treaty of Munster and Treaty of Osnabrück (Treaty of Westphalia), in which Mazarin's policies were French rather than Catholic and brought Alsace (though not Strasbourg) to France; he settled Protestant princes in secularized bishoprics and abbacies in reward for their political opposition to Austria. In 1658 he formed the League of the Rhine, which was designed to check the House of Austria in central Germany. In 1659 he made peace with Habsburg Spain in the Peace of the Pyrenees, which added to French territory Roussillon and northern Cerdanya — as French Cerdagne — in the far south as well as part of the Low Countries.

Towards Protestantism at home, Mazarin pursued a policy of promises and calculated delay to defuse the armed insurrection of the Ardèche (1653), for example, and to keep the Huguenots disarmed: for six years they believed themselves to be on the eve of recovering the protections of the Edict of Nantes, but in the end they obtained nothing.

Towards the pontificate of the successful Spanish candidate, Cardinal Pamphilj, elected pope (15 September 1644) as Innocent X (Cardinal Mazarin having arrived too late to present the French veto), there was constant friction. Mazarin protected the Barberini cardinals, nephews of the late pope, and the Bull against them was voted by the Parliament of Paris "null and abusive"; France made a show of preparing to take Avignon by force, and Innocent backed down. Mazarin was more consistently an enemy of Jansenism, in particular during the formulary controversy, more for its political implications than out of theology. On his deathbed he warned young Louis "not to tolerate the Jansenist sect, not even their name."

Mazarin was not liked by ordinary Frenchmen. In Paris in 1648, popular discontent erupted into open violence. Paris was a city of about half a million people in the mid-seventeenth century. In 1644, Mazarin tried to prevent it growing further and to raise taxes by fining those who built houses outside the City Walls. This policy produced widespread resentment. The Fronde began in January 1648, when the Paris mob used children's slings, frondes, to hurl stones at the windows of Mazarin's associates. Mazarin's continual need to raise money for the war against the Habsburgs provoked the troubles known as the Fronde of the Parlement. Mazarin proposed that the magistrates of the high courts forego their salaries for a number of years; they were outraged, as was the parliament of Paris, because although its deputies' salaries were not threatened, Mazarin wanted to create new offices that would undermine its powers. The Parlement joined with other government bodies to demand various reforms. These included suppressing the intendants, reducing taxation, and forbidding all new taxes without the consent of the parliament, no imprisonment without trial, and limiting the creation of new offices of state. Anne and Mazarin responded by ordering the arrest of several deputies of the parliament, including the popular Pierre Broussel. The Paris mob rioted and built barricades in the streets, forcing the release of Broussel and the others. Renewed disturbances in Paris led Anne to take Louis and leave Paris, in March 1649, the government confirmed the Declaration of October, in return for which Paris and the Parlement laid down their weapons and allowing royal troops to return. Anne and Mazarin did not yet consider it safe for themselves or the king to return.

Many frondeurs had been unhappy with the compromise reached in 1649 and one of their leaders, Jean François Paul de Gondi, had been trying for some time to recruit Louis II de Bourbon, Prince de Condé to their cause. Mazarin feared that an alliance between Condé and the Fronde was imminent. On 18 January 1650 Mazarin had Condé, his brother Armand de Bourbon, prince de Conti and his brother-in-law, Henri II d'Orléans, duc de Longueville arrested. The agreements of 1649 had brought peace to Paris, but there was unrest in other parts of France where supporters and opponents of the government raised forces and disrupted tax collection and administration. The arrest of Condé provoked these areas to open revolt, as Condé's friends and allies spread out across the country recruiting forces to oppose Mazarin and liberate the princes. Condé's wife raised a revolt in Bordeaux, while his sister, and Henry de la Tour d’Auvergne, Viscount of Turenne raised troops and sought Spanish help against the government. Mazarin and Anne were strong militarily, but when the Condéans, the Fronde and the parlement allied and demanded the princes' release, their political position collapsed. In February 1651, Anne freed the princes while Mazarin, fearing the parliament's vengeance, fled the country. The Prince of Condé unfortunately, although a fine general, was an incompetent politician, who soon alienated nobles, parlement, and Parisians. In the Fall of 1651, Condé openly revolted against the crown. In July 1652 his troops entered Paris, but acted with such brutality that his cause lost credibility.

Although in exile, Mazarin had not been idle and had reached agreement with Turenne, a general as talented as Condé. Turenne's forces pursued Condé's, who in 1653 fled to the Spanish Netherlands. Louis XIV, now of age to claim his throne, re-entered Paris in October 1652 and recalled Mazarin in February 1653. The last vestiges of resistance in Bordeaux fizzled out in the late summer of 1653. The French people suffered terribly in the Fronde, but it achieved no constitutional reform. Royal absolutism was reinstalled without any effective limitation.

Death found him seated in his chair, dressed in his full cardinal's robes, and his beard carefully trimmed, as if for a levée; he continued to sign dispatches while his hand could grasp a pen; power passed away only with life. To the last he was consistent with his old hypocrisy; a few hours before his decease he sent a message to the Parliament, in which he declared that its very humble servant died. The event took place on 9 March 1661.

The same day he died, Louis XIV received the dispensation from the then Pope Alexander VII regarding the marriage of Philippe de France, Monsieur and his first wife Princess Henrietta Anne of England.

Cardinal Mazarin's wealth (he collected benefices and amassed a huge fortune and a greater collection of art than the king's) and his nieces' beauty, made for notable family connections, marital and extramarital. His three nieces Hortense, Marie, and Olympia, were famous for their wit, their beauty and their freedom. Olympia was the mother of the famous Prince Eugene of Savoy. Hortense was also a mistress of Charles II of England. Another niece Laura married Alfonso IV d'Este, Duke of Modena and was the mother of Mary of Modena, Queen of England. Altogether, his seven nieces were referred to as the Mazarinettes.