The line crosses a total of eight municipalities: Karlsruhe, Stutensee, Graben-Neudorf, Waghäusel, Neulußheim, Hockenheim, Oftersheim, Schwetzingen and Mannheim.

The geographical situation of the abandoned halt of Altrip, which was on the boundary of the city of Mannheim, is unusual; Altrip lies on the opposite side of the Rhine in the state of Rhineland-Palatinate, having been cut off by the straightening of the river, while the Rhine railway is exclusively located within Baden-Württemberg.

However, the indirect route via Heidelberg bypassed part of the natural catchment area of Mannheim which was located close to the Rhine, including towns such as Schwetzingen and Hockenheim.

This led to a vigorous debate about the southern part of a proposed line from Mannheim to Karlsruhe close to the Rhine.

The Grand Duke of Baden, Frederick I approved the building of a railway via Linkenheim, Eggenstein and Neureut to Mühlburger Tor (to the west of central Karlsruhe) and from there run on the Maxau Railway (Maxaubahn) to the old Karlsruhe central station (this route is now called the Hardt Railway, Hardtbahn), rather than a proposed route running further east to Karlsruhe on the current alignment.

It was taken over by the Grand Duchy of Baden State Railway (Großherzogliche Badische Staatsbahn), on 4 August 1870, the day of its opening.

The now bypassed Karlsruhe–Eggenstein–Graben-Neudorf section was subsequently called the Hardt Railway and converted into a connecting line.

The passenger trains at this time mainly used Silberling carriages, hauled by class 141 electrics locomotives.

In 1997, Karlsruhe-Hagsfeld station, which had been disused since the 1980s, was reactivated in connection with the opening of the Karlsruhe–Blankenloch section of the Karlsruhe Stadtbahn.

Since 2000, the line has been served by Regional-Express services every two hours, running from Karlsruhe via Speyer, Ludwigshafen and Worms to Mainz.

With the start of regular S-Bahn operations, all intermediate stations will be modernised and their platforms will be raised.