Bourbon commanded the troops of Holy Roman Emperor Charles V in what became known as the Sack of Rome in 1527, where he was killed.

[2] However, Francis was uneasy with the proud and wealthy duke, and soon recalled him from Milan and refused to honor his debts.

Suzanne had left all her estates to Charles, but the king's mother, Louise of Savoy, claimed them as the heir in proximity in blood due to their previous entailments.

The emperor gave Charles command of a mixed Spanish–German army (which included a number of Lutherans) sent to chastise Pope Clement VII.

The death of Charles—the artist and goldsmith Benvenuto Cellini claimed that he fired the shot that killed him—outside the walls removed the last restraints from the army, which resulted in the sack of Rome.

However, the county of Montpensier and dauphinate of Auvergne (but not the duchy of Bourbon) were later returned to his sister Louise, who was married to Louis, Prince of La Roche-sur-Yon, a member of the more junior House of Bourbon-Vendome.