In 1496, Joanna, at the age of sixteen, was betrothed to Philip the Handsome, Duke of Burgundy (titular), in the region of Flanders in the Low Countries. Philip's parents were Maximilian I, Holy Roman Emperor and his first wife, Duchess Mary of Burgundy. The marriage was one of a set of family alliances between the Habsburgs and the Trastámaras designed to strengthen both against growing French power.
Joanna entered a proxy marriage at the Palacio de los Vivero in the city of Valladolid, Castile (her parents had secretly married there in 1469). In August 1496 Joanna left from the port of Laredo in northern Spain on the Atlantic's Bay of Biscay. She would not see her mother or siblings again except for her younger sister Catherine of Aragon in 1506, who was then the Princess Dowager of Wales. She would see her father Ferdinand II again in the course of his ruthless efforts to prevent her and Philip from gaining and then from keeping their crowns.
Joanna began her journey to Flanders in the Low Countries, which consisted of parts of the present day Netherlands, Belgium, Luxembourg, France, and Germany, on 22 August 1496. The formal marriage took place on 20 October 1496 in Lier, north of present day Brussels. Between 1498 and 1507, she gave birth to six children; two emperors and four queens.
Joanna's life with Philip was rendered extremely unhappy by his infidelity and her political insecurity. He consistently attempted to usurp her legal birthrights to power. This led in no small part to rumors of her insanity, stoked by reports of her depressive or neurotic acts while she was imprisoned or manipulated by her husband. Before her mother's death in 1504, husband and wife were already living apart.
The death of Joanna's brother John, the stillbirth of John's daughter and the deaths of Joanna's older sister Isabella and Isabella's son Miguel made Joanna heiress to the Spanish kingdoms. Her remaining siblings were Maria (1482–1517) and Catherine (1485–1536), younger than Joanna by three and six years, respectively.
On 25 September 1506 Philip died suddenly of typhoid fever in the city of Burgos in Castile. Some suspected that he had been poisoned by his father-in-law Ferdinand II who had always disliked his foreign Habsburg origins and with whom he never wanted to share power. Joanna was pregnant with their sixth child, a daughter Catherine (1507–1578). By 20 December 1506 she was in the village of Torquemada in Castile, attempting to exercise her rights to rule alone in her own name as Queen of Castile. The country fell into disorder. Her son and heir-apparent, Charles, later Charles I, was a six-year old child being raised in his aunt's care in northern European Flanders; her father, Ferdinand II, remained in Aragon, allowing the crisis to grow. A regency council under Archbishop Cisneros was set up against the queen's orders but it was unable to manage the growing public disorder; plague and famine devastated the kingdom with supposedly half the population perishing of one or the other. The queen was unable to secure the funds required to assist her to protect her power. In the face of this, Ferdinand II returned to Castile in July 1507. His arrival coincided with a remission of the plague and famine, a development which quieted the instability and left an impression that his return had restored the health of the kingdom.