Purpose: The zona incerta (ZI) is below the ventral tier of the thalamus and has a strong influence selectively in higher-order thalamic relays. Although neuropathic pain has been suggested to result from reduced gamma-aminobutyric acid (GABA) and GABAergic signaling in the ZI, the mechanisms remain unclear. Here, the role of GABA and GABAergic signaling was investigated in the ZI in neuropathic pain using sciatic nerve chronic constriction injury (CCI) rats. Materials and methods: Single-unit neuronal activity was recorded, and microdialysis was performed in the ZI of CCI rats and sham-treated rats in vivo. This study also compared ZI neuronal activity after treatment with saline, the GABA_A receptor agonist (muscimol), or the GABA_A receptor antagonist (bicuculline). Results and conclusion: CCI rats exhibited hypersensitivity to pain as evidenced by decreased hind paw withdrawal threshold and latency. CCI rats also showed reduced GABA level and decreased neuronal activity in the ZI compared with sham-treated rats. Treatment with GABA_A receptor agonist, but not GABA_A receptor antagonist, ameliorated pain hypersensitivity and increased the firing rate (spikes/s) of ZI neurons in CCI rats.