Until 1895, the old route of the Odenwald Railway from Obrigheim connected before the line reaches the current site of the Mosbach station, which was built in 1997.

The line leaves the Neckar Valley, running along the Elz and Auerbach rivers, to enter an area of karst landscape known as the Bauland.

The line runs through Adelsheim tunnel to the Kirnau valley, where it connects with the Franconia Railway in Osterburken.

In 1854 the Ludwig's Western Railway was opened from Bamberg in Bavaria to the Hessian border near Kahl am Main through Würzburg.

Both Bavaria and Baden saw reasons to build a railway line between Heidelberg and Würzburg: the government of Baden sought a continuation of its main line to the north east and the Bavarian king, Maximilian II, sought a connection with the Palatinate, then a geographically separate part of Bavaria.

Instead, after a review of 45 alternative routes and lengthy negotiations, a decision was taken to build a line through Meckenheim, Mosbach, Osterburken and Lauda.

Duplication of the line together with the Neckar Valley Railway between Neckargemünd and Neckarelz began in 1904 and was completed on 29 April 1906.

During the air war over Germany the line—as opposed to the Neckarelz railway junction—was not a prime target of Allied attacks.

After the war operations on the railway between Neckarelz and Osterburken were resumed in June 1945, as the line had great importance as a supply route for the occupation of the American zone.

When, in the early 1970s, Deutsche Bundesbahn electrified the Neckar Valley Railway and the Bad Friedrichshall-Jagstfeld–Würzburg line with financial support from the state of Baden-Württemberg, it was decided to electrify the gap between Neckarelz and Osterburken as well, allowing the continuous electrical operation of freight trains on the Rhine-Neckar–Franconia route.

The line had lost its importance for passenger traffic as an east–west route as a result of the division of Germany at the end of the war.

Since the clearance of the bridge of the Mosbach-Mudau narrow-gauge railway was insufficient for electrification, this meant the end of the line, which was already threatened with closure.

After disagreements between the city of Mosbach and Deutsche Bahn over the transfer of the site of the old station for the upgrading of the B 27, an agreement was reached in 1994 over the use of unused infrastructure.

The planning for the Rhine-Neckar S-Bahn network originally envisaged only a service at 30 minutes intervals along the Neckar Valley Railway to Eberbach.

The offer to extend the S-Bahn to Neckarelz, together with an extension from Speyer to Germersheim was accepted at an initial cost of €75 million.