Human brain imaging studies suggest that chronic neuropathic pain has a strong emotional component that is mediated by medial prefrontal cortex (mPFC) activity; in rodents, the mPFC is involved in emotional and cognitive aspects of behavior, including the extinction of Pavlovian fear conditioning. Together, these findings suggest that the cortex may modulate the memory trace of pain. As D-cycloserine (DCS), a partial agonist of the NMDA receptor, can enhance learning and potentiate the extinction of acquired fear, in the present study we tested its efficacy in neuropathic pain behavior. In rats with spared nerve injury (SNI), repeated daily oral administration of DCS reduced mechanical sensitivity of the injured limb in a dose-dependent manner; this effect continued for weeks after the cessation of DCS treatment. In addition, re-exposure to DCS further enhanced antinociceptive behavior. Repeated oral DCS administration also reduced cancer chemotherapy drug-induced neuropathic pain behavior. Infusions of DCS directly into the mPFC (especially within prelimbic cortex) or the amygdala (but not into thalamus, insula, or occipital cortex) acutely induced antinociception in SNI rats. The antinociceptive effect of intra-mPFC DCS infusions was mimicked by NMDA and glycine, and blocked by HA 966. In the mPFC of SNI rats, NR2B expression was down-regulated; however, this effect was reversed with repeated oral DCS. Lastly, infusions of DCS into mPFC reversed place avoidance behavior induced by mechanical stimulation of the injured paw in SNI rats. These findings indicate that limbic NMDA-mediated circuitry is involved in long-term reduction in neuropathic pain behavior.