The monastery was founded in 1364, but nothing remains of its first wooden structures. The significant flourishing of the monastery is associated with Vasily III, who in the early 16th century donated substantial funds for the construction of the Protection Cathedral and the Holy Gates, as well as, presumably, the cells and the fence, which have not survived to this day. One of the first inmates of the monastery was Vasily III's wife, Solomonia Saburova, who was tonsured as a nun under the name Sophia in 1525 due to accusations of infertility.

Later, a legend emerged that Solomonia was pregnant at the time of her tonsure and gave birth in the monastery to Tsarevich Georgy, who allegedly grew up to become the bandit Kudejary. In 1934, restorer A. D. Varganov opened the tomb of Solomonia's supposed son and found only a child's shirt there. However, historian Dmitry Volodikhin claims that the protocol for the tomb opening is missing, and excavations in 1996 showed that burial was impossible due to lack of space.

In the monastery's burial vault under the Protection Cathedral, the names of noble inhabitants have been preserved, such as Ivan III's daughter Alexandra, Ivan IV's wife Anna Vasilchikova, Vladimir Staritsky's first wife Evdokiya Nagaya, Yevpraksia Pozharskaya, and Tsarevna Anastasia. The widow of Vasily Shuisky, Queen Maria Buynosova-Rostovskaya, was also imprisoned here before moving to the Novodevichy Monastery.

In 1551, after the death of Ivan the Terrible's daughter, the Zachatyevskaya refectory church was rebuilt, replacing the wooden structure from the 14th century. In the 17th century, an apse was added, a kitchen was built, and the construction of the fence continued.

In 1923, the monastery was closed and devastated. From 1933, a military biological laboratory was located within its walls, where imprisoned specialists developed biological weapons. In 1934, it was transferred to the biochemistry institute of the Red Army. In 1935, Nikolai Gaysky and Boris Elbert created the world's first liquid vaccine against tularemia. The laboratory operated until 1936.

In the 1950s-1960s, the monastery buildings were restored and became museum exhibitions. In the 1980s, a tourist hotel complex with a restaurant and bar was established here, and a concert hall was located in the cathedral.

Since 1992, monastic life has been revived in the monastery, and it has become active again. It owns the cathedral, the almshouse building, and economic structures. The cells were rebuilt in the 1970s. On April 22, 2020, the Vladimir diocese received the command hut for free use, the last building not previously transferred to the monastery.