The present study investigated the protective effects of melatonin (MT) against gentamicin (GM)-induced nephrotoxicity and oxidative stress in rats. We also investigated the effects of MT on induction of apoptotic cell death and its potential mechanisms in renal tissues in response to GM treatment. The following four experimental groups were evaluated: (1) vehicle control, (2) MT (15 mg/kg/day), (3) GM (100 mg/kg/day), and (4) GM&MT. GM caused severe nephrotoxicity as evidenced by increased serum blood urea nitrogen and creatinine levels, increased renal tubular cell apoptosis, and increased Bcl2-associated X protein and cleaved caspase-3 protein expression. Additionally, GM treatment caused an increase in levels of inducible nitric oxide synthase (iNOS) and nuclear factor-kappa B (NF-κB) protein expression in renal tissues. The significant decreases in glutathione content, catalase, superoxide dismutase, glutathione-S-transferase, glutathione peroxidase, and glutathione reductase activities and the increase in malondialdehyde content indicated that GM-induced tissue injury was mediated through oxidative reactions. In contrast, MT treatment protected kidney tissue against the oxidative damage and the nephrotoxic effect caused by the GM treatment. Histopathological studies confirmed the renoprotective effect of MT. These results indicate that MT prevents nephrotoxicity induced by GM in rats, presumably because it is a potent antioxidant, restores antioxidant enzyme activity, and blocks NF-κB and iNOS activation in rat kidney.