Vincenzo was the son of Duke Vincent I and Eleonora de' Medici[2] and inherited the duchy upon the death of his elder brother Ferdinand, receiving the imperial investiture on 8 February 1627.

He received a cardinalate in 1615 upon Ferdinando's succession, but did not travel to Rome to receive the red hat;[3] the following year he married his relative Isabella Gonzaga, daughter of Alfonso Gonzaga, Count of Novellara, and the pope deprived him of his cardinalate.

He and Isabella did not have any children, and as the only surviving son, he was encouraged to take a new wife; however the pope would not grant him a divorce.

[4] Vincenzo also had problems with money and sold several of his paintings and valuables, particularly to the English King Charles I.

[4] Vincenzo II Gonzaga had no legitimate offspring from his wife, but he recognized four natural sons.