Vesicle associated membrane protein 2 (VAMP2), also called synaptobrevin-2, is known to play a critical role in neurotransmitter release associated with SNARE-mediated exocytosis. Recently, VAMP4, another member of VAMP family proteins, has been shown to participate in GABA release mediated by a different exocytotic mechanism. We investigated subcellular distribution and function of VAMP4 in hippocampal neurons during inhibitory synapse development. Hippocampal neurons under primary culture established from embryo of vesicular GABA transporter (VGAT)-Venus transgenic mice were transiently transfected with adenovirus expressing VAMP2-mCherry and VAMP4-mTurquoise2. Confocal imaging showed that VAMP2 was mainly localized in the presynaptic varicosities of GABAergic neurons starting from an early stage of synapse formation. In contrast, VAMP4 started to occur in the varicosities at a later stage during synapse maturation. To further examine the effects of overexpressed VAMP2 or VAMP4 on inhibitory neurotransmission, we recorded stimulation-evoked GABAergic synaptic currents in cultured pyramidal neurons during synapse formation and maturation. VAMP4 overexpression caused an increase in GABA release probability following tetanic stimulation, and this effect could be observed at a later stage of synapse development, but not at an early stage. Our data indicate that presynaptic VAMP4 is involved in GABAergic neurotransmission in parallel to inhibitory synapse maturation.