The late Romanesque basilica was first mentioned in 1239. In 1398, it was replaced by a large church building with a nave constructed in the typical style of the late Gothic period. The chancel was completed by 1410, and the building of the nave took until 1441.

There was also a delay in the construction of the church tower, which was started again in 1508. The chancel contained the tombs of the Prince Electors of the Palatinate. Most of the memorial tablets were destroyed in 1693. Of the originally 54 epitaphs, the only one to remain was the one of Prince Elector Ruprecht II (1352 - 1410).

During the Thirty Years War, however, the complete collection became the booty of Heidelberg's conqueror, Commander Tilly. Following orders of the Catholic Prince Elector, Maximilian of Bavaria, the library was given to the Pope of Rome. Of the collection, which once included more than 5,000 books and 3524 manuscripts, only 885 manuscripts were returned to Heidelberg in 1816.

During the following centuries, the church frequently changed its religious denomination and was used at different times by Catholics as well as Protestants. Even a partition barrier was erected in 1706 because both denominations wanted to hold service here. For 230 years, the barrier stayed in its place until it was removed in 1936. Today, the Church of the Holy Spirit is a Protestant Church.