Background: The neocortex, including the medial prefrontal cortex (mPFC), contains many neurons expressing nitric oxide synthase (NOS). In addition, increasing evidence shows that the nitric oxide (NO) and opioid systems interact in the brain. However, there have been no studies on the interaction of the opioid and NO systems in the mPFC. The objective of this study was to investigate the effects of administrating L-arginine (L-Arg, a precursor of NO) and N(gamma)-nitro-L-arginine methyl ester (L-NAME, an inhibitor of NOS) into the mPFC for neuropathic pain in rats. Also, we used selective opioid receptor antagonists to clarify the possible participation of the opioid mechanism. Methods: Complete transection of the peroneal and tibial branches of the sciatic nerve was applied to induce neuropathic pain, and seven days later, the mPFC was cannulated bilaterally. The paw withdrawal threshold fifty percent (50% PWT) was recorded on the 14th day. Results: Microinjection of L-Arg (2.87, 11.5 and 45.92 nmol per 0.25 μL) increased 50% PWT. L-NAME (17.15 nmol per 0.25 μL) and naloxonazine (an antagonist of mu opioid receptors, 1.54 nmol per 0.25 μL) inhibited anti-allodynia induced by L-Arg (45.92 nmol per 0.25 μL). Naltrindole (a delta opioid receptor antagonist, 2.45 nmol per 0.25 μL) and nor-binaltorphimine (a kappa opioid receptor antagonist, 1.36 nmol per 0.25 μL) were unable to prevent L-Arg (45.92 nmol per 0.25 μL)-induced antiallodynia. Conclusions: Our results indicate that the NO system in the mPFC regulates neuropathic pain. Mu opioid receptors of this area might participate in pain relief caused by L-Arg.