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Pain is one of the most common medical challenges, reducing life quality. Despite the progression in pain management, it has remained a clinical challenge, which raises the need for investigating novel antinociceptive drugs with correspondence signaling pathways. Besides, the precise antinociceptive mechanisms of melatonin are not revealed. Accordingly, owing to the critical role of l-arginine/nitric oxide (NO)/cyclic GMP (cGMP)/K ATP in the antinociceptive responses of various analgesics, the role of this signaling pathway is evaluated in the antinociceptive effects of melatonin. Male NMRI mice were intraperitoneally pretreated with the injection of l-arginine (NO precursor, 100 mg/kg), N (gamma)-nitro-l-arginine methyl ester [l-NAME, NO synthase (NOS) inhibitor, 30 mg/kg], S-nitroso-N-acetylpenicillamine (SNAP, NO donor, 1 mg/kg), sildenafil (phosphodiesterase inhibitor, 0.5 mg/kg), and glibenclamide (K ATP …