The reformers Martin Luther and Johannes Bugenhagen preached there and the building also saw the first celebration of the mass in German rather than Latin [dubious – discuss] and the first ever distribution of the bread and wine to the congregation [dubious – discuss] – it is thus considered the mother-church of the Protestant Reformation.

In 1996, it was inscribed on the UNESCO World Heritage List along with Castle Church of All Saints (Schlosskirche), the Lutherhaus, the Melanchthonhaus, and Martin Luther's birth house and death house in Eisleben, because of its religious significance and testimony to the lasting, global influence of Protestantism.

Between 1412 and 1439 the nave was replaced by the present three-aisle structure and the two towers built, originally crowned by stone pyramids.

In 1522, in the wake of the iconoclasm begun by Andreas Bodenstein, almost the whole interior decoration was demolished and removed, leaving the still-surviving High Medieval Judensau on the exterior of the south wall.

Luther married Katharina von Bora here on 13 June 1525, the service being conducted by his colleague and friend, Johannes Bugenhagen.

From 1533 to 1817 the Stadtkirche's pastor was also general superintendent of the Saxon Electoral Circle (Kurkreis) and thus granted to the top theological lecturer at the University of Wittenberg.

In 1988, on the occasion of the 50th anniversary of the Kristallnacht, debate sprung up about the monument, which resulted in the addition of a sculpture recognizing that during the Holocaust six million Jews were murdered "under the sign of the cross".