Padilla was a member of an ancient noble family of Toledo. Charles, who came to the Spanish throne in 1516, had inflamed national opinion by appointing to high posts foreigners who carried out arbitrary and exploitative actions. Demands soon arose for the imposition of traditional Castilian constitutional checks on royal power. Padilla had personal grievances against Charles as well and took part in dissident activities in Toledo in late 1519 and early 1520. Summoned in April 1520 to appear before the king at Santiago, Padilla instead took up arms in support of a popular uprising in Toledo.

A circular letter from Toledo to other Castilian cities in revolt invited them to meet at Avila. When the municipalities, supported by the nobles and clergy, set up the Junta Santa (a revolutionary junta) there in July 1520, Padilla was named captain general of its forces, and on August 29 he took Tordesillas, thereby assuring the junta's control over Charles's mother, the hereditary queen Joan the Mad, who had been living there since she had gone insane in 1506.

The junta soon alienated the nobility by its popular demands, and Charles cleverly moved to secure the nobility's loyalty. The junta also courted defeat in the field by replacing Padilla with Don Pedro Girón, an important nobleman. After Charles's troops had recovered Tordesillas (December 5) and Girón had defected, the Junta Santa recalled Padilla. Padilla's reappointment was received with a great outpouring of popular enthusiasm. He occupied Torrelobatón on February 28, 1521. Seven weeks later, however, on the advance of royal forces, he attempted to retreat but was defeated and captured at Villalar (April 23, 1521). He was executed the next day along with other leaders of the revolt.