We investigated the antinociceptive effect of microinjection of HSV-1 amplicon vector-mediated human proenkephalin (hPPE) into the ventral periaqueductal grey (PAG) on neuropathic pain in rats. Male Sprague-Dawley rats with chronic constriction injury (CCI)-induced neuropathic pain were microinjected into the ventral PAG with normal saline (NS), pHSVIRES-lacZ (SHZ), or HSV-1 amplicon vector pHSVIRES-hPPE-lacZ (SHPZ), respectively. Pain thresholds in the SHPZ-treated rats were significantly higher at day 3, then reached peak at day 14 and lasted until day 35 after PAG administration, and these effects were reversed by naloxone. In contrast, NS or SHZ-treated rats did not significantly affect pain thresholds. These results demonstrated that microinjection of HSV-1 amplicon vector-mediated hPPE into the ventral PAG attenuates neuropathic pain in rats.