The cathedral was Algirdas's gift to Uliana, and a useful diplomatic gesture toward the growing Christian populations of his largely pagan duchy. By the early fifteenth century, between 1415 and 1795, it served as the mother church of the Ruthenian Orthodox Church and, after the 1596 Union of Brest, the Ruthenian Uniate Church across the Grand Duchy of Lithuania and the Polish-Lithuanian Commonwealth. Helena of Moscow, the Orthodox daughter of Ivan III of Muscovy and wife of the Lithuanian Grand Duke Alexander Jagiellon, was buried inside in 1513. After Lithuania converted to Roman Catholicism in 1387, the Orthodox cathedral survived in part because of the patronage of two Ruthenian princes, Konstanty Ostrogski and Konstanty Wasyl Ostrogski, who restored it after the dome collapsed in 1506. After their line ended, the Uniate Church took control in 1609 and rebuilt it in the regional Catholic style.

A major fire in 1748 emptied the building. For decades it sat partially abandoned, used for various secular purposes. A baroque reconstruction in 1785 restored some of its function as a church, but during the Kosciuszko Uprising of 1794 the Russian army shelled and destroyed it again. By 1808, with the building beyond easy repair and the local clergy unwilling to fund restoration, a Vilnius prelate sold the ruin to Vilnius University. The university overhauled what was left of the cathedral in 1822 in the Neoclassical style under the architect Karol Podczaszynski. For roughly half a century, the medieval Orthodox foundation housed an anatomy theater where students dissected cadavers, a university library, and assorted teaching facilities. It is one of the more peculiar afterlives in European church history: a fourteenth-century Orthodox sanctuary spending the early industrial era as a place where lecturers held up specimens for medical students.

After the failed January Uprising of 1863, the Russian Empire imposed a sweeping Russification campaign on Lithuania and Poland. Count Mikhail Nikolayevich Muravyov, called the Hangman of Vilnius for his suppression of the uprising, and his brother led the campaign in this region. They engineered the confiscation of the old cathedral building from the university and its transfer back to the Russian Orthodox Church. The architect Nikolai Chagin, brought from Russia for the work, rebuilt the cathedral between 1865 and 1868 in a style imitating medieval Georgian church architecture. The choice was political. Reasserting an Orthodox cathedral in central Vilnius, in a style meant to evoke ancient Christian authority, was a way of signaling that Russian Orthodoxy belonged here, regardless of what the local Catholic and Jewish populations might think. The reconstructed cathedral functioned as the seat of the Russian Orthodox Diocese of Lithuania.

St. Tikhon, born Vasily Belavin, served as Archbishop of Vilnius from 1913 until the German occupation of 1915. He would later become Patriarch of Moscow and All Russia, the first to be elected after the abolition of the patriarchate by Peter the Great two centuries earlier; he was canonized by the Russian Orthodox Church in 1989. When German troops took Vilnius in 1915, most of the Russian Orthodox clergy retreated east with the imperial army. The cathedral remained but its parish was sharply diminished. Damaged again during the Second World War, it was restored in 1948 and renovations were finally completed in 1957. A further restoration in 1998 brought the building closer to its nineteenth-century form.

Today the cathedral belongs to the Russian Orthodox Diocese of Lithuania. Its services are attended mostly by ethnic Russians and Belarusians who live in Vilnius, communities that account for roughly 12 percent of the city's population. The building sits in the Old Town, a UNESCO World Heritage site, near the Vilnia River and Gediminas Tower. The political weight of a Russian Orthodox cathedral in the capital of a Baltic state, particularly after 2022, is considerable. The Lithuanian Orthodox Church has sought partial autonomy from the Moscow Patriarchate; some clergy have publicly broken with Moscow's positions on Ukraine. The Cathedral of the Theotokos, the oldest surviving piece of Christian architecture in Vilnius, contains within its stones every layer of that history: a pagan duke's gift to his Christian wife, a university's anatomy classroom, an empire's Russification monument, and a working parish church for people whose religious identity is now politically charged.