Objective: Methotrexate (MTX) leads to acute toxic side effects in tissues or organs containing rapidly dividing cells such as seminiferous tubules. In this study, we investigated the protective effects of vitamin C against MTX-induced injury in rat testis. Material and methods: A total of 31 rats were divided into 4 groups, including the control group. The study was completed within 4 weeks and the rats received daily doses of 2 mL/kg SF, 100 mg/kg vitamin C and 10 mg/kg/day MTX i.p according to their groups. The mean seminiferous tubular diameter, germinal epithelial cell thickness, and mean testicular biopsy score were determined by histologic examination of each group. Results: The vitamin C + MTX group showed more similarity with the control group. Statistically significant results were achieved between groups as for mean seminiferous tubular diameter, germinal epithelial cell thickness, and mean testicular biopsy score. When compared with the group which received vitamin C after MTX therapy, values for mean seminiferous tubular diameter, germinal epithelial cell thickness, and mean testicular biopsy score were significantly higher in the group which received vitamin C before initiation of MTX therapy. Conclusion: Vitamin C decreased MTX-induced testicular histological injuries, especially when used before MTX therapy.