On Federico's death in 1540, their eldest son,
Francesco became
Duke of Mantua as well as Marquis of Montferrat. As Francesco was still only a minor aged eight, Margaret acted as his regent. Margaret had her son married to
Catherine, daughter of
Ferdinand I, Holy Roman Emperor and
Anna of Bohemia and Hungary. Margaret had her second son,
Guglielmo married to Catherine's younger sister,
Eleanor. Francesco died one year after his marriage to Catherine in 1550, and no children were born to their union. Margaret's second son Guglielmo then became Duke of Mantua. Margaret acted as his regent, with the help of her brother-in-law Cardinal
Ercole Gonzaga. As regent, Margaret issued laws and regulations which benefitted the economy of Mantua by the establishment of businesses. She made improvements on the river harbour and had the fortifications and defence of the Duchy strengthened. In her foreign policy, she was pro-Spanish, which caused the French king to occupy Montferrat in 1555 – it was to be held by France until the Treaty of Cambresis in 1559. Her regency was terminated when her son came of age in 1556. Margaret died in
Mantua on 28 December 1566. In 1574, Margaret's homeland of
Montferrat became part of the Duchy of Mantua, after the death of Margaret's son. In Montferrat, Guglielmo was known as
Guglielmo X. ==Issue==