Jacques Cartier set sail for a second voyage on May 19 of the following year with three ships, 110 men, and the two natives. Reaching the St. Lawrence, he sailed up-river for the first time, and reached the Iroquoian capital of Stadacona, where Chief Donnacona ruled. Jacques Cartier left his main ships in a harbour close to Stadacona, and used his smallest ship to continue up-river and visit Hochelaga (now Montreal) where he arrived October 2, 1535. Hochelaga was far more impressive than the small and squalid village of Stadacona, and more than 1,000 Iroquoians came to the river edge to greet the Frenchmen. The site of their arrival has been confidently identified as the beginning of the Sainte-Marie Sault - where the bridge named after him now stands. The expedition could proceed no further, as the river was blocked by rapids. So certain was Cartier that the river was the Northwest Passage and that the rapids were all that was preventing him from sailing to China, that the rapids and the town that eventually grew up near them came to be named after the French word for China, La Chine: the Lachine Rapids and the town of Lachine, Quebec.

After spending two days among the people of Hochelaga, Cartier returned to Stadacona on October 11. It is not known exactly when he decided to spend the winter of 1535 - 1536 in Stadacona, and it was by then too late to return to France. Cartier and his men prepared for the winter by strengthening their fort, stacking firewood, and salting down game and fish. During this winter, Cartier compiled a sort of gazetteer that included several pages on the manners of the natives — in particular, their habit of wearing only leggings and loincloths even in the dead of winter.... From mid November 1535 to mid April 1536, the French fleet lay frozen solid at the mouth of the St. Charles River, under the Rock of Quebec. Ice was over a fathom (1.8 m) thick on the river, with snow four feet (1.2 m) deep ashore. To add to the discomfort, scurvy broke out — first among the Iroquoians, and then among the French. In his journal, Cartier states that by mid February, "out of 110 that we were, not ten were well enough to help the others, a pitiful thing to see". Cartier estimated the number of natives dead at 50. One of the natives who survived was Dom Agaya, the chief's son who had been taken to France the previous year. During a friendly visit by Domagaya to the French fort, Cartier enquired and learned from him that a concoction made from a tree known as annedda (probably arbor vitae) would cure scurvy. This remedy likely saved the expedition from destruction, allowing 85 Frenchmen to survive the winter.

Ready to return to France in early May 1536, Cartier decided to take Chief Donnacona to France, so that he might personally tell the tale of a country further north, called the "Kingdom of Saguenay", said to be full of gold, rubies and other treasures. After an arduous trip down the St. Lawrence and a three-week Atlantic crossing, Cartier and his men arrived in Saint-Malo on July 15, 1536, concluding the second, 14 month voyage, which was to be Cartier's most profitable.

On October 17, 1540, Francis I ordered the Breton navigator to return to Canada to lend weight to a colonization project of which he would be "captain general". However, January 15, 1541 saw Cartier supplanted by Jean-François de La Rocque de Roberval, a Huguenot courtier and friend of the king named as the first lieutenant general of French Canada. Roberval was to lead the expedition, with Cartier as his chief navigator. While Roberval waited for artillery and supplies, he gave permission to Cartier to sail on ahead with his ships.

On May 23, 1541, Cartier departed Saint-Malo on his third voyage with five ships. This time, any thought of finding a passage to the Orient was forgotten. The goals were now to find the "Kingdom of Saguenay" and its riches, and to establish a permanent settlement along the St. Lawrence River. Anchoring at Stadacona, Cartier again met the Iroquoians, but found their "show of joy" and their numbers worrisome, and decided not to build his settlement there. Sailing a few miles up-river to a spot he had previously observed, he decided to settle on the site of present-day Cap-Rouge, Quebec. The convicts and other colonists were landed, the cattle that had survived three months aboard ship were turned loose, earth was broken for a kitchen garden, and seeds of cabbage, turnip, and lettuce were planted. A fortified settlement was thus created and was named Charlesbourg-Royal. Another fort was also built on the cliff overlooking the settlement, for added protection. The men also began collecting what they believed to be diamonds and gold, but which upon return to France were discovered to be merely quartz crystals and iron pyrites, respectively — which gave rise to a French expression: "faux comme les diamants du Canada" ("As false as Canadian diamonds"). Two of the ships were dispatched home with some of these minerals on September 2.

Having set tasks for everyone, Cartier left with the longboats for a reconnaissance in search of "Saguenay" on September 7. Having reached Hochelaga, he was prevented by bad weather and the numerous rapids from continuing up to the Ottawa River. Returning to Charlesbourg-Royal, Cartier found the situation ominous. The Iroquoians no longer made friendly visits or peddled fish and game, but prowled about in a sinister manner. No records exist about the winter of 1541 - 1542 and the information must be gleaned from the few details provided by returning sailors. It seems the natives attacked and killed about 35 settlers before the Frenchmen could retreat behind their fortifications. Even though scurvy was cured through the native remedy (Thuja occidentalis infusion), the impression left is of a general misery, and of Cartier's growing conviction that he had insufficient manpower either to protect his base or to go in search of the Saguenay Kingdom.

Cartier left for France in early June 1542, encountering Roberval and his ships along the Newfoundland coast, at about the time Roberval marooned Marguerite de La Rocque. Despite Roberval's insistence that he accompany him back to Saguenay, Cartier slipped off under the cover of darkness and continued on to France, still convinced his vessels contained a wealth of gold and diamonds. He arrived there in October, in what proved to be his last voyage. Meanwhile, Roberval took command at Charlesbourg-Royal, but it was abandoned in 1543 after disease, foul weather and hostile natives drove the would-be settlers to despair.

Cartier spent the rest of his life in Saint-Malo and his nearby estate, where he often was useful as an interpreter in Portuguese, and he died aged 65 or 66 on September 1, 1557 from an epidemic. No permanent European settlements were made in Canada before 1608, when Samuel Champlain founded Quebec City. Cartier is interred in St. Vincent's Cathedral.

Having already located the entrance to the St. Lawrence on his first voyage, he now opened up the greatest waterway for the European penetration of North America. He produced an intelligent estimate of the resources of Canada, both natural and human, albeit with a considerable exaggeration of its mineral wealth. While some of his actions toward the St. Lawrence Iroquoians were dishonourable, he did try at times to establish friendship with them and other native peoples living along the St. Lawrence River — an indispensable preliminary to French settlement in their lands.

Cartier was the first to document the name Canada to designate the territory on the shores of the St Lawrence River. The name is derived from the Huron-Iroquois word "kanata", or village, which was incorrectly interpreted as the native term for the newly discovered land. Cartier used the name to describe Stadacona, the surrounding land and the river itself. And Cartier named "Canadiens" the inhabitants (Iroquoians) he had seen there. Thereafter the name Canada was used to designate the small French colony on these shores, and the French colonists were called Canadiens, until the mid-nineteenth century, when the name started to be applied to the loyalist colonies on the Great Lakes and later to all of British North America. In this way Cartier is not strictly the European discoverer of Canada as this country is understood today, a vast federation stretching a mari usque ad mare (from sea to sea). Eastern parts had previously been visited by the Norse, as well as Basque, Galician and Breton fishermen, and perhaps the Corte-Real brothers and John Cabot (in addition of course to the Natives who first inhabited the territory). Cartier's particular contribution to the discovery of Canada is as the first European to penetrate the continent, and more precisely the interior eastern region along the St. Lawrence River. His explorations consolidated France's claim of the territory that would later be colonized as New France, and his third voyage produced the first documented European attempt at settling North America since that of Lucas Vázquez de Ayllón in 1526-27.

Cartier's professional abilities can be easily ascertained. Considering that Cartier made three voyages of exploration in dangerous and hitherto unknown waters without losing a ship, and that he entered and departed some 50 undiscovered harbors without serious mishap, he may be considered one of the most conscientious explorers of the period. Cartier was also one of the first to formally acknowledge that the New World was a separate land mass from Europe/Asia.

On August 18, 2006, Quebec Premier Jean Charest announced that Canadian archaeologists had discovered the precise location of Cartier's lost first colony of Charlesbourg-Royal. The colony was built where the Cap Rouge river runs into the St. Lawrence River and is based on the discovery of burnt wooden timber remains that have been dated to the mid-16th century, and a fragment of a decorative Istoriato plate manufactured in Faenza, Italy, between 1540 and 1550, that could only have belonged to a member of the French aristocracy in the colony. Most probably this was the Sieur de Roberval, who replaced Cartier as the leader of the settlement. This colony was the first known European settlement in modern day Canada since the c.1000 AD L'Anse aux Meadows Viking village in northern Newfoundland. Its rediscovery has been hailed by archaeologists as the most important find in Canada since the L'Anse aux Meadows rediscovery.