He was the second son of Gustav Vasa (1523 – 1560). His mother was Margareta Leijonhufvud (1514 – 1551), a Swedish noblewoman. As a Duke of Finland he opposed his half - brother Eric XIV of Sweden (1560 – 1568) and was imprisoned in 1563. After his release from prison, probably because of his brother's insanity, John again joined the opposition, deposed Eric and made himself the king. His important ally was his maternal uncle Sten Leijonhufvud, who at deathbed was made Count of Raseborg. Shortly after this John executed his brother's most trusted counsellor, Jöran Persson, whom he held largely responsible for his harsh treatment while in prison.

John finished the Scandinavian Seven Years' War in 1570 without many Swedish concessions and during the following years he successfully fought Russia in the Livonian War, concluded by the Treaty of Plussa in 1583, a war that meant a Swedish reconquest of Narva. As a whole his foreign policy was affected by his connection to Poland of which country his son Sigismund III Vasa was made king in 1587.

In domestic politics John showed clear Catholic sympathies, inspired by his Polish queen, a fact that created frictions to the Swedish clergy and nobility. He launched the "Red Book" which reintroduced several Catholic customs. In 1575, he gave his permission for the remaining Catholic convents in Sweden to start receiving novices again. From time to time he was also at odds with his younger brother Duke Charles of Sudermannia (afterwards Charles IX of Sweden). John III was an eager patron of art and architecture.

In January of 1569, John was recognized as king by the same riksdag that forced Eric XIV off the throne. But this recognition was not without influence from John; Duke Karl received confirmation on his dukedom without the restrictions of his power that the Arboga articles imposed. The nobilities' power and rights were extended and their responsibilities lessened.

John was still concerned about his position as king as long as Eric was alive. The fear of a possible liberation of the locked up king worried him to the point that in 1571 he ordered the guards that in the event of any suspicion of liberation attempt to murder the captured king. It is possible this is how his life ended in 1577.

John III was reportedly like his father in propaganda, with repeated claims to have "liberated Sweden" from the "bloodhound" Christian II, as well as rescuing the population from the "tyrant" Eric XIV. He was reportedly violent, hot tempered and greatly suspicious.

John married his first wife, Catherine Jagellonica of Poland (1526 – 1583), house of Jagiello, in Vilnius on 4 October 1562. In Sweden she is known as Katarina Jagellonica and she was the sister of king Sigismund II Augustus of Poland. They had three children. He married his second wife Gunilla Bielke (1568 – c. 1592) on 21 February 1584 and together they had one son. Together with his mistress Karin Hansdotter (1532 – 1596) he had at least four illegitimate children.

John cared for Karin and his children with her after he married with Catherine Jagellonica of Poland in 1562. He took care that Karin got a husband that should take care of her and his children. In 1561 Karin was married to a nobleman Klas Andersson (Västgöte), a friend and servant of John. Together they had a daughter named Brita. When John became King in 1568 he continued to support Karin and his illegitimate children. In 1572 Karin was married again, her earlier husband was murdered by Erik XIV in 1563, to a Lars Henrikson. In 1576 he was ennobled by John for taking care of his issue with Karin. The same year he sent for his daughter Sofia to be a lady in the castle as a servant to his sister Princess Elisabet.