From the life of St. Pafnuty, it is known that the new stone church, built on the site of the former wooden church, was painted by iconographers Mitrofan and Dionysius. The cathedral was so magnificent that even the Moscow princes admired it. The white-stone church lasted just over a hundred years.

In the late 16th century, by the order and funds of Tsar Fyodor Ivanovich, a new brick church was built to replace the old white-stone one, consecrated in 1589 and named the Church of the Nativity of the Most Holy Theotokos. The five domes of the cathedral, designed in a helmet style, gave it severity and grandeur. The altar was initially separated by a brick wall, which was later replaced by an iconostasis. In the southern part of the altar, behind the brick wall, a chapel was arranged in honor of St. Theodore Stratilates, and a church in honor of St. Irene the Great Martyr adjoined the northern wall. These names were chosen in honor of the heavenly patrons of Tsar Fyodor and Tsarina Irina, sister of Boris Godunov. The crosses on the cathedral are adorned with royal crowns, emphasizing the tsar's patronage.

It is known that Fyodor Ivanovich visited this place for prayer in 1592 and 1595. Over 400 years, the cathedral underwent numerous changes. In the mid-17th century, porches were built on three sides, and later the windows were altered. In the 17th century, domes were added to replace the helmet-shaped endings, so that the cathedral's height matched the new tall bell tower. In 1837, a brick refectory was added to the back wall, which has survived to this day. The difference in their construction is 250 years. During the restoration, it was decided to preserve and update the refectory to trace changes in architectural forms. The annex, executed in late classicism, is the latest structure of the monastery. In 1997-1998, a chapel was arranged in it in honor of St. Pafnuty. The cathedral can be entered from the southern and northern sides.

In the past, there were frescoes on the walls of the cathedral, but by the mid-19th century, they had only survived above the southern and northern portals under iron canopies. Inside, the cathedral is adorned with Byzantine-style iconography. The lower part of the northern and southern walls of the Church of the Nativity of the Theotokos contains a cycle of the life of St. Pafnuty Borovsky. The frescoes were covered with oil twice, but some original scenes have survived in the chapel of St. Theodore Stratilates. In the 20th century, after the monastery was returned to the Church, restorers managed to restore the frescoes.

In the 1920s, the cathedral was used as a storage hangar, which gradually deteriorated. In 1954, the central drum collapsed, damaging two agricultural grain harvesters, as the monastery housed an agricultural training institution. During the restoration, which began in 1962, it was found that the cause of the destruction was foundation deformation. Work was temporarily halted due to strange incidents, and the area near the altar remained untouched.

The vaults of the central dome and four side domes are supported by four pillars. The cathedral is equipped with a system of voice pipes to enhance acoustics. The largest voice pipe is located in the northwestern pillar. There is a doorway in the northern wall leading to a hiding place under the roof, where valuables were stored.

There is a legend of two underground passages from the cathedral to Kaluga and Moscow. Monks from the Pafnutiev Monastery were allowed to trade in Moscow and Kaluga, and they could reach there through underground passages, avoiding worldly temptations. The floors in the cathedral are now stone with heating, but they were previously made of iron plates.