Developing a successful treatment strategy for neuropathic pain has remained a challenge among researcher and clinicians. Various animal models have been employed to understand the pathogenic mechanism of neuropathic pain in experimental animals. The present study was designed to explore the possible nitric oxide mechanism in the protective effect of melatonin against chronic constriction injury (CCI) of sciatic nerve in rats. Following chronic constriction injury, various behavioral tests (thermal hyperalgesia, cold allodynia) and biochemical parameters (lipid peroxidation, reduced glutathione, catalase, and nitrite) were assessed in sciatic nerves. Drugs were administered for 21 consecutive days from the day of surgery. CCI significantly caused thermal hyperalgesia, cold allodynia and oxidative damage. Chronic administration of melatonin (2.5 or 5 mg/kg, ip) significantly attenuated hyperalgesia, cold allodynia and oxidative damage in sciatic nerves as compared to CCI group. Further, L-NAME (5 mg/kg) pretreatment with sub-effective dose of melatonin (2.5 mg/kg, ip) significantly potentiated melatonin's protective effect which was significant as compared to their individual effect per se. However, L-arginine (100 mg/kg) pretreatment with melatonin (2.5 mg/kg, ip) significantly reversed its protective effects. Results of the present study suggest the involvement of nitric oxide pathway in the protective effect of melatonin against CCI-induced behavioral and biochemical alterations in rats.