In the 1990s the nature of the line changed fundamentally with the establishment of the Rhine-Neckar S-Bahn.

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 platform height of 76 cm allowed for the operation of the S-Bahn's electric multiple units from the beginning.

At the timetable change 2003/2004 in December 2003, lines S1 and S2 of the Rhine-Neckar S-Bahn were officially opened.

Mosbach (Baden) station is located at kilometer 53.4 of the Neckarelz–Osterburken railway and is served by S-Bahn trains operated by the Rhine-Neckar S-Bahn (line S1: Osterburken–Heidelberg–Mannheim–Homburg (Saar) and line S2: Mosbach (Baden)–Heidelberg–Mannheim–Kaiserslautern).