Background/aim: Methotrexate (MTX), used commonly as an antimetabolite drug in cancer therapy, leads to acute toxic side effects in tissues or organs containing rapidly dividing cells, such as bone marrow, gastrointestinal mucosa, and seminiferous tubules. In this study, we investigated the protective effects of vitamin E and L-carnitine against MTX-induced injury in rat testis.Materials and methods: Rats were divided into 4 groups, including the control group. The study took 17 days and the animals received daily doses of 0.5 mL/kg saline, 250 mg/kg vitamin E, or 500 mg/kg L-carnitine intraperitoneally. The experimental groups received 20 mg/kg methotrexate intraperitoneally on days 3 and 10.Results: Weight loss, testicular weight loss and marked histological injuries, increased malondialdehyde levels, and decreased superoxide dismutase levels were only observed in the MTX-treated groups. Vitamin E and L-carnitine treatments did not affect MTXinduced weight loss or testicular weight loss, but they inhibited MTX-induced testicular histological injuries. Vitamin E and L-carnitine treatments suppressed the increases in malondialdehyde levels and the decreases in superoxide dismutase levels. Conclusion:Vitamin E and L-carnitine treatments decreased MTX-induced testicular histological injuries, and these results were supported by biochemical measurements.