Born at Chaumont-sur-Loire into the House of Amboise, Charles was the nephew of Cardinal Georges d'Amboise, prime minister of King Louis XII of France, and the son of Charles I d'Amboise, governor of Champagne and Bourgogne. Charles d'Amboise acted as governor of Paris, of the Duchy of Milan, of the seignory of Genoa, and of the province of Normandie. In 1501 he was made French lieutenant general and then vice-roy for Lombardy, becoming a friend of Leonardo da Vinci during his stay in Milan. He was Grand Master of France from 1502 to 1504, when he became Marshal of France. From 1508 to 1510 he was also admiral of France and in 1507 suppressed a revolt in Genoa.