The monastery was founded in 1650 by the abbot of the Mhar Monastery, Callistratus, with the support of the Kyiv Metropolitan Sylvester Kosiv and the funds of the first Poltava colonel, Martyn Pushkar, as well as other representatives of the Cossack elite and townspeople. In 1695, the monastery was burned by the Crimeans, and in 1709, the Swedes, placing their cannons here, shelled the city. According to legend, Charles XII was wounded in the leg near the monastery, which limited his mobility on the day of the Battle of Poltava. In memory of the victory, starting from 1803, the archimandrites of the monastery were instructed to wear images of St. Sampson the Hospitable and St. George the Victorious on their mantles. In 1857, the Holy Sampson Church was annexed to the monastery on the battlefield.

From 1775 to 1786, the monastery served as the residence of the Slavic bishops, and from 1786 to 1797, of the Ekaterinoslav bishops. The famous Ekaterinoslav Archbishop Ambrose Serebrennikov, known for his sermons and piety, was buried here in 1792, as well as St. Athanasius Volkovsky in 1801. In the 1790s, the vicar of the Ekaterinoslav diocese, Bishop Ioasaph Potemkin, lived in the monastery. From 1884, the monastery became the residence of the vicar bishop of Pryluky. The monastery was closed in 1923, revived as a women's monastery in 1942, closed again in 1960, and revived once more in 1991.

The history of the Slavic seminary began at the monastery, created through the efforts of Archbishop Eugene Bulgaris. In 1778, a school for training monastery singers was opened here, which soon transformed into a theological school and was moved to the city, where it became a seminary in 1780. Before the seminary was relocated to Novomyrhorod in 1798, notable figures such as Ivan Kotliarevsky, Mykola Hnatyshyn, and Vasyl Hohol-Yanovsky studied here. The seminary was under the administration of the Slavic bishops, and its rectors were Archbishop Nikifor Theotokis and Metropolitan Gabriel Banulesco-Bodoni. From 1818, a male theological school existed at the monastery, which was located in a building on the hillside until 1876, when it was moved to the city.

The territory of the monastery is elongated from north to south in the shape of a narrow rhombus with truncated vertices. It is surrounded by a brick fence built in the first half of the 1990s. In the center of the northern part stands a 47-meter bell tower from 1786 in the style of Ukrainian Baroque, reminiscent of the Great Bell Tower of the Kyiv-Pechersk Lavra. To the west of the bell tower are the Holy Gates. North of the bell tower, outside the monastery, a memorial sign was erected in 1991 in honor of the destroyed necropolis of the monastery during the Soviet era. South of the bell tower are the Exaltation Cathedral, the Holy Trinity Church, and the Church of St. Simeon the God-receiver.

The Exaltation Cathedral was built at the turn of the 17th and 18th centuries in the style of Ukrainian Baroque, combining elements of ancient Russian, wooden Ukrainian, and Western architecture. Between the bell tower and the cathedral lies the grave of Sister Antonina Zhartovska, who gathered signatures for the revival of the monastery. Between the cathedral and the Holy Trinity Church is a rotunda over a revered spring.

The Holy Trinity Church was built from 1995 to 1999 on the site of the destroyed during the Soviet era church of the same name from the 18th-19th centuries, which served as a burial place for archbishops. In 2001, Metropolitan Theodosius Dykun of Poltava and Kremenchuk was buried in the crypt of the church.

The Church of St. Simeon the God-receiver was erected in 1887 by the Poltava philanthropist Innocent Kotelnikov in memory of his only son, Simeon, who died in Poltava on his way south for treatment of tuberculosis. Before his death, the boy bequeathed to be buried in the Exaltation Monastery, which he liked very much.

Along the western part of the monastery fence, opposite the cathedral, is the building of cells, built in 1770 and renovated in 1863 and 1892. South of it, opposite the Holy Trinity Church, which has parish status, a house for the parish Holy Trinity community was built in the 1990s. Further south, along the fence, stretches the building of cells constructed in 1889 by Innocent Kotelnikov, with a domestic church of St. Seraphim of Sarov built in the first half of the 1990s. In the southern and eastern parts of the monastery estate, there are also several utility buildings.