He was born in Antwerp, the son of leading Antwerp painter Quinten Matsys and the older brother of Cornelis, who became a painter and engraver. He trained under his father. He was admitted, together with his brother Cornelis, as a master in the Antwerp Guild of St. Luke in 1531, a year after their fathers death. It is assumed that he left Antwerp immediately thereafter and worked for a while in Fontainebleau, but these facts are not firmly established. He was back in Antwerp by 1536. He married his cousin Anna van Tuylt in 1538. The couple would have three children.
In 1544 Jan and his brother Cornelis were banned from Antwerp because of their religious beliefs. It is possible that Jan went to Fontainebleau and Germany. It is certain that he spent time in Genoa. He returned to Antwerp before the end of 1555 when the ban imposed on him was ended. He was then involved in a number of court cases with his brothers and sisters over the distribution of inheritances.