The septohippocampal (SH) pathway comprises cholinergic and GABAergic fibers. Whereas the former establish synaptic contacts with all types of hippocampal neurons, the latter form complex baskets specifically on interneurons. The GABAergic SH function is associated with the control of hippocampal synchronous networks. Little is known about the mechanisms involved in the formation of the GABAergic SH pathway. Semaphorin (Sema) 3C is expressed in most hippocampal interneurons targeted by these axons. To ascertain whether Sema 3C influences the formation of the SH pathway, we analyzed the development of this connection in Sema 3C-deficient mice. As these animals die at birth, we developed anin vitroorganotypic co-culture model reproducing the postnatal development of the SH pathway. In these SH co-cultures, the GABAergic SH pathway developed with target specificity similar to that presentin vivo. SH axons formed incipient baskets on several types of hippocampal interneurons at 7 daysin vitro, which increased their complexity by 18–25 daysin vitro. These SH fibers formed symmetric synaptic contacts on GABAergic interneurons. This synaptic specificity was not influenced by the absence of entorhinal afferents. Finally, the absence of Sema 3C in target neurons or its blockage by neuropilin-1 and -2 ectodomains in slice co-cultures did not lead to major changes in either the target specificity of the GABAergic SH pathway or its density of innervation. We conclude that the formation and synaptic specificity of the GABAergic SH pathway relies on robust molecular mechanisms, independent of Sema 3C, that are retained in ourin vitroco-culture model.