After the mysterious death of William II in August 1100, his brother, Henry, immediately seized the royal treasury and crown. His next task was to marry and Henry's choice was Matilda. Because Matilda had spent most of her life in a convent, there was some controversy over whether she was a nun and thus canonically ineligible for marriage. Henry sought permission for the marriage from Archbishop Anselm, who returned to England in September 1100 after a long exile. Professing himself unwilling to decide so weighty a matter on his own, Anselm called a council of bishops in order to determine the canonical legality of the proposed marriage. Matilda testified that she had never taken holy vows, insisting that her parents had sent her and her sister to England for educational purposes, and her aunt Cristina had veiled her to protect her "from the lust of the Normans." Matilda claimed she had pulled the veil off and stamped on it, and her aunt beat and scolded her for this act. The council concluded that Matilda was not a nun, never had been and her parents had not intended that she become one, giving their permission for the marriage.

Matilda and Henry seem to have known one another for some time before their marriage — William of Malmesbury states that Henry had "long been attached" to her, and Orderic Vitalis says that Henry had "long adored" her character.

Her mother was the sister of Edgar the Ætheling, proclaimed but uncrowned King of England after Harold, and through her, Matilda was descended from Edmund Ironside and thus from the royal family of Wessex, which in the 10th century, had become the royal family of a united England. This was very important as Henry wanted to make himself more popular with the English people and Matilda represented the old English dynasty. In their children, the Norman and English dynasties would be united. Another benefit was that England and Scotland became politically closer; three of her brothers became kings of Scotland in succession and were unusually friendly towards England during this period of unbroken peace between the two nations: Alexander married one of Henry I's illegitimate daughters and David lived for some time before his accession at Henry's court.  Most of her dower estates were granted from lands previously held by Edith of Wessex.

After Matilda and Henry were married on 11 November 1100 at Westminster Abbey by Archbishop Anselm of Canterbury, she was crowned as "Matilda," a fashionable Norman name. By courtiers, however, she and her husband were soon nicknamed 'Godric and Godiva'.  These two names were typical English names from before The Conquest and mocked their more rustic style, especially when compared to the flamboyance of William II.

She gave birth to a daughter, Matilda, in February 1102, and a son, William, called "Adelin", in November 1103. As Queen, she maintained her court primarily at Westminster, but accompanied her husband on his travels around England, and, circa 1106–1107, probably visited Normandy with him. Matilda was the designated head of Henry's curia and acted as regent during several of his absences.

During the English investiture controversy (1103-07), she acted as intercessor between her husband and archbishop Anselm. She wrote several letters during Anselm's absence, first asking him for advice and to return, but later increasingly to mediate.