Methotrexate, a well-known cytotoxic chemotherapy, anti-folate, immunosuppressant, effectively treats all major disorders like cancer, psoriasis, refractory rheumatoid arthritis, etc. Abnormal production of reactive oxygen species has been suspected in the pathophysiology of methotrexate-induced renal toxicity. Vitamin C is a water soluble antioxidant that can play a protective role in models of experimentally induced nephropathies. This study aimed to clarify the protective role of vitamin C on the histological and biochemical changes induced by methotrexate in renal cortex. Twenty adult rats were divided into four equal groups. In group I, the rats received a balanced diet for 9 days; in group II, the rats received 250mg/kg B.w of vitamin C for 9 days by oral gavage dissolved in distilled water; in group III, the rats received a single dose of 20mg/kg B.w of methotrexate intraperitoneally on the third day of the experiment; in group IV, the rats received a single dose of 20mg/kg B.w of methotrexate intraperitoneally on the third day of the experiment and 250mg/kg B.w of vitamin C by oral gavage 2 days before, and 6 days after methotrexate administration. Results: methotrexate-induced renal injury in rats was evidenced biochemically by the significant increase in serum levels of creatinine and urea as well as histopathological alterations. Furthermore, methotrexate administration caused apoptosis by increasing activity of caspase-3. Furthermore, vitamin C reduced renal oxidative stress, and prevented the alterations in renal morphology. Conclusion: methotrexate produces marked degenerative changes in the histological structure of the kidney, and vitamin C is a potent antioxidant agent in preventing kidney injury.