She 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 was the fourth of their five surviving children, and had a stillborn twin sister named Anna.
As an infanta of Spain, her hand in marriage was very important in European politics; before her marriage to Manuel I of Portugal, her parents entertained the idea of marrying her off to King James IV of Scotland. This was at a time when her younger sister Catherine's marriage to Arthur, Prince of Wales, was being planned. Ferdinand and Isabella thought if Maria was Queen of Scotland, the two sisters could keep the peace between their husbands. These plans, however, came to nothing. Her eldest sister Isabella, Princess of Asturias, was the first wife of Manuel I, but her death in 1498 created a necessity for Manuel to remarry; Maria became the next bride of the Portuguese king, reaffirming dynastic links with Spanish royal houses.
Manuel and Maria were married in Alcácer do Sal on 30 October 1500, and was granted Viseu and Torres Vedras as her dower. She had 10 children, eight of whom reached adulthood, including King John III of Portugal, Holy Roman Empress Isabella, and Beatrice, Duchess of Savoy.
Queen Maria became known as a fervent Catholic. 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. She co-founded the Jeronimos Monastery in Lisbon.
Maria died in Lisbon on 7 March 1517, and was buried at the Jerónimos Monastery of Belém. She died as a result of her last pregnancy.