The Rab3 GDP/GTP exchange protein (Rab3 GEP) activates the Rab3 small GTP-binding protein (G protein) family, including Rab3A that is an important member controlling synaptic vesicle trafficking. Here, we examined the role of Rab3 GEP in regulating neurotransmitter release in autapses of mouse hippocampal neurons in culture. The release probability was markedly reduced in Rab3 GEP-/- neurons, whereas the readily releasable pool size was not different between WT and Rab3 GEP-/- neurons, indicating that Rab3 GEP up-regulates a postdocking step of synaptic exocytosis. Because Rab3A reportedly down-regulates Ca(2+)-triggered fusion of synaptic vesicles, these results provide evidence for a role of Rab3 GEP in the postdocking process distinct from Rab3A activation.