She served as regent of Spain for her brother Philip II during his trips to England to marry Mary I from 1554 to 1556, and 1556 to 1559.

Later that year Joanna returned to Spain at her father's request, leaving her young son in the care of her mother-in-law, who was also her aunt.

Joanna returned to Spain in May 1554 at the request of her father, leaving her newborn son with her mother-in-law, the Portuguese Queen Catherine of Austria, who was Charles V's youngest sister.

Shortly after Sebastian's birth, Joanna was called back to Madrid by her brother Philip to act as regent during his absence in England from 1554.

In 1555, she is reputed to have been admitted surreptitiously to the male-only Jesuit order under the name of a pseudonym, Mateo Sánchez.