Cyclophosphamide (CP), an alkylating antineoplastic agent, has potential urotoxicity including causing hemorrhagic cystitis (HC). HC is now accepted as a non-infectious inflammation and the pathogenesis of HC includes cytokine production which leads to inducible nitric oxide synthase (iNOS) induction. Moreover, overproduction of reactive oxygen species (ROS) during inflammation leads to extensive oxidative stress, cellular injury and apoptosis/necrosis via several mechanisms. Based on these facts, the aim of this study was to evaluate the protective effects of melatonin as an antioxidant, iNOS inhibitor and peroxynitrite scavenger against CP-induced urinary bladder damage. A total of 30 male Sprague-Dawley rats were divided into four groups. Three groups received a single dose of CP (100 mg/kg) intraperitoneally with the same times. Group 2 received CP only, group 3 received 5 mg/kg/day and group 4 received 10 mg/kg/day melatonin before and the day after CP administration. Group 1 served as the control. Increased iNOS induction, bladder malonyldialdehyde (MDA) levels and urinary nitrite-nitrate excretion were encountered in the CP-only group leading to severe cystitis. Melatonin exhibited significant protection against CP-induced cystitis by diminishing bladder oxidative stress and blocking iNOS and peroxynitrite production. Oxidants may have a major role in the pathogenesis of CP-induced cystitis and iNOS is an important mediator leading to peroxynitrite production. Melatonin ameliorates bladder damage induced by CP.