In 1363, Sergius of Radonezh blessed the Novgorod monks Feodor and Pavel to establish a monastery. The legend states that Sergius chose the site for the new monastery, predicting its prosperity: "God and the Most Holy Mother of God will bless this place, and Boris and Gleb will help, elevating the monastery." All the buildings were originally wooden, and the monastery existed in this form for 159 years.

The Borisoglebsky Monastery enjoyed special favor from the Moscow princes and the first Russian tsars, who considered it their "home". During the internecine wars of the 15th century, Grand Duke Vasily II the Dark found refuge here. In 1440, the abbot Pitirim baptized Ivan III, the son of Vasily the Dark, in the monastery. Ivan III, like his father, generously endowed the monastery. Under his son, Vasily III, stone construction began in 1522. For this, master Grigory Borisov was invited. The monastery established brick production, extracted sand and lime, and brought boulders for the foundations. Ivan IV the Terrible visited the monastery three times, first in 1545, and made over 20 donations, including books, money, and a bell weighing 138 poods. Donations were also made by Tsars Boris Godunov, Alexei Mikhailovich, and other noble families. By the mid-17th century, the monastery owned 22,000 desyatinas of land and four subordinate monasteries, making it one of the wealthiest in the Rostov Metropolis. In the early 17th century, the venerable Irinarh labored here, who predicted to Vasily Shuisky the "captivity of Russia by the Poles". The monastery was visited by military leaders and princes, who saw in Irinarh a spiritual mentor.

In the late 17th century, simultaneously with the construction of the Rostov Kremlin, large-scale construction work began in the monastery on the orders of Metropolitan Ion Sysoevich. However, the secularization of church income and land under Peter I and Catherine II turned the monastery into an ordinary second-class one. In the 18th-19th centuries, the monastery existed on 280 desyatinas of land, a garden, and income from renting towers and shops. Among the ruling figures, only Catherine II and Alexander III visited here, and even then only in passing.

The monastery was officially abolished in 1924. Some buildings were transferred to the Rostov State Museum, while others remained with the believers. By the end of 1929, there were no functioning churches left. In 1989, services resumed in the Cathedral of Boris and Gleb, and in December 1994, the Borisoglebsky Men's Monastery was revived. Since 1924, a branch museum of the State Museum-Reserve "Rostov Kremlin" has been operating on its territory, featuring exhibitions and open access to the fortress walls and observation tower.