He was born in Florence, the second son of Cosimo I de' Medici, Grand Duke of Tuscany, and Eleanor of Toledo.

Aged only sixteen, after having already been made Archbishop of Pisa, Giovanni was created cardinal of Santa Maria in Domnica, by Pope Pius IV in the consistory of 31 January 1560.

Probably already suffering from tuberculosis, Giovanni died in Livorno two years after he was made a cardinal, from an attack of malaria.

Centuries after his death, a myth arose to the effect that Garzia had killed Giovanni, following a dispute in 1562.

In turn, their father Cosimo was supposed to have killed Garzia in a rage with his own sword.