The Church of Panagia Acheiropoietos is located in the central part of the walled city of Thessalonica, a short distance to the north of Egnatia Street. It is mentioned in sources as the large church of the Virgin Mary; the name Acheiropoietos is first attested in a document from 1320, doubtless in connection with a devotional icon of the Virgin Mary at prayer, believed to have been created without human hand, which was kept in the church. It appears that in Byzantine times the Virgin Mary was venerated together with the city's patron saint, Agios Demetrius, and that on the latter’s feast day the litany entered the Church of the Virgin Mary as its principal station, before proceeding to the Church of Agios Demetrius. The church was built after 450, in the third quarter of the 5th century. In 1430, immediately after the fall of Thessalonica to the Ottomans, Acheiropoietos was the first church to be converted into a mosque, which conquest is recorded in a Turkish inscription on the eighth column in the north colonnade , counting from the east.

From an architectural point of view, Acheiropoietos is a three-nave timber roofed basilica with galleries . The three naves in the main church are separated by colonnades, while a tribelon opening leads from the narthex to the nave. The north nave ends to the east in the Middle Byzantine Chapel of Agia Irini. To the east, the central nave ends in a semicircular conch with a synthronon and episcopal throne. The triple-light window with pillars replaced the original five-light window with mullions . The marble altar screen is modern; traces on the floor reveal that the original one ended at the third column from the east. To the west is the narthex, with visible traces of an exonarthex , which perhaps correspond to the east portico of the atrium that would have been extended to the west. On the south side of the building there is a monumental propylon , probably because the church was connected to the city’s main thoroughfare, known as the Leophoros or Avenue. An outbuilding next to the propylon is believed to have been the baptistry . The three successive floors in the north nave of the basilica belong to an earlier Roman bath.

Over the course of its history the monument has undergone a series of alterations, distorting its original form to some degree.  The most serious of these include destruction of the original roof, degradation of the upper part of the central nave (leading to the loss of the skylight), and demolition of the west gallery and exonarthex.

Thanks to the robustness and external grandeur of the basilica, the harmony and balance of its internal layout, the high artistic merit of its mosaic and sculpture decoration and its marble floor, the church is considered one of the most important examples of its kind in the whole of Greece.

The sculpture decoration forms a unified whole designed and executed for this particular basilica. Of particular importance are the mid-5^th century Theodosian capitals , which share features with those from Constantinople, and the Thessalian marble columns in the tribelon.

The high quality mosaics in the colonnade intrados on the ground floor and south gallery , the narthex and the window of the west wall should be assigned to the same period in; they depict crosses, water vessels, birds, fruit, fish and other themes with religious symbolism, in symmetrical arrangement. The sponsor of the mosaics is referred to as Andrew, on the intrados of the central and south arc of the tribelon. He has been identified as the priest who took part in the Council of Chalcedon (451) as representative of the Archbishop of Thessalonica, which fits in with the dating of the mosaics to the second half of the 5th century.

All that survives from the Byzantine period are a few poorly preserved murals in the south nave, on the wall above the south colonnade. The eighteen figures alternately shown in profile and full face form part of a depiction of the Forty Martyrs of Sebaste, which dates to the early decades of the 13th century.

The church has undergone repeated renovation work since the early 20th century, including drastic alterations to the supporting structure and extensive rebuilding of the damaged or dilapidated sections.  Recently completed large-scale maintenance and restoration projects were aimed at repairing the damage caused to the monument by the 1978 earthquake, and guaranteeing the monument’s overall structural stability.