Maria was born at Córdoba on 29 June 1482 as the third surviving daughter of Isabella I of Castile and Ferdinand II of Aragon (the Catholic monarchs).

She had 10 children, eight of whom reached adulthood, including King John III of Portugal, Holy Roman Empress Isabella, and Beatrice, Duchess of Savoy.

She maintained a close correspondence with her parents, got along well with her sisters-in-law the duchess Isabella of Braganza and the queen dowager Eleanor, and her mother-in-law Beatrice, and hosted a large court with both Spanish and Portuguese ladies-in-waiting.

She supported King Manuel's religious-imperial project, including the plan to conquer the Mamluk's realm, destroy Mecca and Medina and reconquer Christian holy places such as Jerusalem.

This state of affairs resulted in a continual deterioration of her health and after the delivery of 1516, she was reportedly exhausted to a point that she was also temporarily mentally confused before she recuperated.