Information on Antonio de' Medici's birth is shrouded in mystery. As a newborn baby he was presented to Francesco I de' Medici (1541-1587) by his mistress Bianca Cappello as the fruit of their relation. The woman simulated her pregnancy and childbirth, and with the help of a compliant physician and maidservant procured the baby from anonymous parties. In spite of the fact that Bianca's deceit was later revealed, Francesco I forgave her, and in 1583 recognized Antonio as his legitimate son. He showed him great affection and brought him up as a Medici.

At his adoptive father's death his uncle and the new Grand Duke of Tuscany Ferdinando I (1549-1609) at first deprived the boy of a large part of his inheritance. Subsequently though, after making sure of his subservience, he restored to him his place at Court, where Antonio continued to live as a prince of the Medici house. As sole condition, he was named Prior of the Jerusalem Knights, who were obliged to celibacy.

Don Antonio took a special interest in the Spagiric art and in the fusion of metals, and carried on his activities at the country house of San Marco, which had been the setting of the alchemical experiences of his putative father. He also took an interest in Galileo's discoveries, and although his correspondence with Galileo was not intense, among the letters the latter received from him there are some of a crucial importance for understanding the evolution of astronomical research.