Thermochronological, structural and kinematic analyses demonstrate the influence of Late Paleozoic basement structures on the localization and evolution of the Rhine–Bresse Transfer Zone (RBTZ), a central segment of the European Cenozoic Rift System. Zircon and apatite fission track data indicate that the Paleozoic crystalline basement and its Permo-Triassic cover, exposed in the La Serre Horst, experienced a Middle Jurassic to Early Cretaceous heating event which was followed by two distinct periods of cooling. A first cooling event occurred in the Early Cretaceous and is probably related to thermal relaxation. A second one, started at around 38 Ma, is interpreted to reflect exhumation that was induced by fault reactivation along the Late Paleozoic Burgundy Trough System during Eocene–Oligocene rifting and the formation of the RBTZ. This fault reactivation resulted in a complex fault pattern and local stress field perturbations in the surroundings of the pre-existing horst structure. Substantial extension was achieved by ENE–WSW striking normal faults that trend highly oblique to the dominant strike of the Rhine and Bresse Graben rifts. Seismic reflection data show that these major normal faults are typically associated with extensional flexures and oriented parallel to pre-existing Late Paleozoic basement faults of the Burgundy Trough. Paleostress analyses yield overall NW–SE extension during the Eo-Oligocene, slightly oblique to the structural trend of the RBTZ, and additionally, local stress field perturbations induced by the reactivation of pre-existing faults. Reactivation of Paleozoic structures in the RBTZ is of extensional rather than of strike-slip character indicating that the RBTZ is forming a separate oblique graben segment within the European Cenozoic Rift System rather than a sinistral strike slip transform zone supposedly connecting Rhine and Bresse graben structures.