Cisplatin (CP) is commonly used in the treatment of various solid tumors. Its use, however, is hampered by nephrotoxicity. In this study, we compared the effect of betaine and melatonin given singly, with that of a combination of these two agents on CP-induced nephrotoxicity in mice. CP (20 mg/kg, given intraperitoneally on the 8th day of 12 days of the experiment) showed the typical physiological, biochemical, and histologic features of nephrotoxicity. CP-treated mice showed a significant reduction in food intake, body weight, and urine and fecal output. It also induced significant increases in the plasma concentrations of urea, creatinine, uric acid, phosphorous, adiponectin, interleukin-1β, interleukin-6, transforming growth factor -β1, tumor necrosis factor-α, and cystatin C. All these effects were significantly reduced by daily administration of betaine or melatonin at oral doses of 200 mg/kg and 10 mg/kg, respectively. Furthermore, using the two agents in combination caused further significant reductions in the above parameters. These findings suggest that betaine and melatonin concomitant use is likely to provide greater protection against CP-induced nephrotoxicity than when they are given singly, rendering them potentially suitable and safe agents to use in clinical trials to assess their possible beneficial actions in cancer patients receiving CP.