Urotoxicity is one of the major problems associated with cyclophosphamide (CP) chemotherapy in cancer patients. Melatonin is a potent antioxidant and reduces CP-induced urotoxicity. However, the molecular mechanisms of protection offered by melatonin are not yet clear. The present study investigated the role of nuclear erythroid 2-related factor 2 (Nrf2) and nuclear factor-kappa B (NF-kappaB) on melatonin-mediated protection against CP-induced urotoxicity. CP was administered intraperitoneally at the dose of 150 mg/kg to induce urotoxicity in male Sprague-Dawley rats. Melatonin treatment (10 mg/kg) was initiated 3 days before and continued for 1 day after the CP administration. Melatonin treatment reduced the CP-induced oxidative stress and DNA damage in the urinary bladder as observed by abrogation in thiobarbituric acid-reactive substances and glutathione levels as well as comet and modified comet assay parameters. Melatonin treatment reduced the bladder damage and apoptosis as observed by histological analysis and TUNEL assay. Melatonin increased the expression of transcription factor Nrf2 as well as associated phase-II enzymes NADPH: quinone oxidoreductase-1 and heme oxygenase-1. Further melatonin treatment reduced the expression of transcription factor NF-kappaB. The results of the present study provide evidence that melatonin treatment favorably alters Nrf2 and NF-kappaB expression and, this appears to be at least in part responsible for observed protection against CP-induced urotoxicity.