Regeneration of synaptic connections between hair cells and spiral ganglion neurons would be required to restore hearing after neural loss. Here we demonstrate by immunohistochemistry the appearance of afferent-like cochlear synapses in vitro after co-culture of de-afferented organ of Corti with spiral ganglion neurons from newborn mice. The glutamatergic synaptic complexes at the ribbon synapse of the inner hair cell contain markers for presynaptic ribbons and postsynaptic densities. We found postsynaptic density protein PSD-95 at the contacts between hair cells and spiral ganglion neurons in newly formed synapses in vitro. The postsynaptic proteins were directly facing the CtBP2-positive presynaptic ribbons of the hair cells. BDNF and NT-3 promoted afferent synaptogenesis in vitro. Direct juxtaposition of the postsynaptic densities with the components of the preexisting ribbon synapse indicated that growing fibers recognized components of the presynaptic sites. Initiation of cochlear synaptogenesis appeared to be influenced by glutamate release from the hair cell ribbons at the presynaptic site since the synaptic regeneration was impaired in glutamate vesicular transporter 3 mutant mice. These insights into cochlear synaptogenesis could be relevant to regenerative approaches for neural loss in the cochlea.