In 1567, the construction of the Vologda Kremlin began, which included plans for a new cathedral on the site of the wooden Resurrection Cathedral, located outside the new city center. In 1568, by order of Ivan the Terrible, the construction of a stone cathedral began on the banks of the Vologda River. Chronicles report that the tsar commanded the construction of the cathedral church of St. Sophia in honor of the Assumption of the Most Holy Theotokos within the city, near the archbishop's residence. However, according to other sources, the Archbishop's residence was moved later, already to the cathedral under construction. Archaeological excavations in 2000 revealed structures in this area dated to the 1560s-1570s.

The architectural model for the cathedral was the Assumption Cathedral in Moscow, as Ivan the Terrible wanted to see a resemblance to the Moscow temple in his northern residence. The tsar personally oversaw the construction and often stayed in Vologda. In 1570, after the main work was completed, he visited the cathedral but soon left the city, and construction was abandoned.

The cathedral remained unfinished until 1587, when under Tsar Fyodor Ioannovich, it began to be completed. Only the southern chapel was finished, while the central part of the temple was completed later. The southern chapel was consecrated in 1587 by Bishop Anthony in honor of the Beheading of John the Baptist, the patron saint of Ivan the Terrible. The chapel was located in the deacon's room of the cathedral until major repairs in 1848-1850.

In 1612, when Vologda was captured by Polish and Lithuanian troops, the cathedral was devastated and damaged by fire. After the invaders left, restoration work began, and in 1612-1613, the cathedral was consecrated again. In 1639, Mikhail Fyodorovich gifted the cathedral a copper chandelier, and later three more chandeliers were acquired in Arkhangelsk.

In 1686-1688, the cathedral was painted by Yaroslavl icon painters under the guidance of Dmitry Grigoriev. Before the painting, the windows were enlarged and small windows were cut into the apses. In 1698 and 1724, the cathedral suffered from fires, after which a new iconostasis was installed in 1737-1744.

Until 1776, services in the cathedral were held year-round. During the repairs from 1848-1851, stone narthexes were added, window openings were enlarged, and the roof was painted. In the 1860s, a stone fence appeared around the cathedral.

The cathedral was visited by emperors: Peter I in 1724, Alexander I in 1824, and Alexander II in 1858. In 1923, the cathedral was closed and transferred to the local history museum, where an exhibition on the history of religion and atheism was placed. Since 1935, the cathedral has been a federal monument of significance.

In 1966-1968, restoration work was carried out under the guidance of architect V.S. Banige, during which the cathedral regained its 17th-century appearance. Inside the temple, ancient frescoes were secured under the guidance of N.V. Pertsev.

From 1999 to 2007, a comprehensive restoration of the cathedral took place. It is now managed by the Vologda State Museum-Reserve and is open for visits in the summer. On certain church holidays, services are held in the cathedral. In June 2007, Patriarch Alexy II performed a solemn liturgy in the cathedral.