Abstract: The perception of pain is altered by inflammatory processes. Anti-inflammatory drugs block this by raising the pain threshold and by reducing the inflammatory process. Melatonin is claimed to have anti-inflammatory activity in animal models of acute and chronic inflammation. However, little is known whether melatonin can reverse the hyperalgesia that is secondary to the inflammation. This study assessed the effect of melatonin on in a well-established model of hyperalgesia associated with inflammation in rats. Peroxynitrite, as generated by the interaction between superoxide anion radical exogenously supplied (O2˙−) and endogenous nitric oxide (NO), led to the development of hyperalgesia. This subplantar injection of O2˙− into the right hindpaw evoked potent thermal hyperalgesia measured by changes in withdrawal latency. Melatonin (25–100 mg/kg, given ip 30 min prior to O2˙−) dose dependently attenuated the hyperalgesic responses to O2˙−. Moreover, melatonin (100 mg/kg) significantly improved tissue damage and inflammation, blocked protein nitration affecting cyclooxygenase-2 and inducible nitric oxide synthase expression in paw tissue. To investigate the antinociceptive activity of melatonin and characterize the underlying mechanisms involved in this action, mitogen-activated protein kinase and NF-κB pathways were explored. Moreover, antihyperalgesic effect of melatonin derived partly from the inhibition of superoxide-driven PARP activation. These results suggest that melatonin has ameliorative potential in attenuating the hyperalgesia associated with inflammation.