Since a sustained higher excitability in the superficial dorsal horn is considered to be a crucial factor leading to chronic pain, normalizing excitatory and inhibitory balance in the dorsal horn by inhibiting GABA transporters (GAT1 and GAT3) is a promising therapeutic strategy for pain relief. However, synaptic mechanisms underlying the analgesic effects of GAT inhibitors remain unknown. Using spinal slice preparations from adult mice, we previously demonstrated that the GAT1 inhibitor NNC-711 decreases the frequency of miniature EPSCs (mEPSCs) in the dorsal horn neurons via presynaptic mechanisms including the activation of GABA_B receptors. In the present study focusing on GAT3 inhibition, we found that the GAT3 inhibitor SNAP-5114 suppressed monosynaptic C-fiber-evoked EPSCs, which was not observed in the presence of the GABA_B receptor antagonist CGP55845. By contrast, A-fiber-evoked EPSCs were less suppressed, and the frequency and the amplitude of mEPSCs were not altered. Thus, although endogenously increased GABA after blockade of GAT1 and GAT3 acts on GABA_B receptors, GAT1 and GAT3 inhibition depresses excitatory neurotransmission from spinal intrinsic interneurons and the primary afferent fibers, respectively.