This study evaluated diosgenin effects on methotrexate-induced testicular injury in the rats. A single dose of methotrexate (MTX) (20 mg/kg, i.p) was administered, followed by two weeks of diosgenin treatment via gavage starting one day before methotrexate injection. Testicular damage was evaluated through histological examination of seminiferous tubules, as well as analysis of serum testosterone level, oxidative stress and inflammation biomarkers, and antioxidant levels. The results of this study showed that in the MTX-exposed group, oxidative stress indices of malondialdehyde (MDA), reactive oxygen species (ROS), nitrite and indices of inflammation consisting of tumor necrosis factor α (TNFα), and interleukin 6 (IL-6) have a significant increase compared to the control group. Additionally, reductions were observed in antioxidant enzymes including superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH). In addition, testosterone level decreased and signs of testicular damage were observed in the MTX group. Conversely, in the group treated with diosgenin alongside MTX at a dosage of 50 mg/kg, there was a significant decrease in oxidative stress markers (MDA, ROS, nitrite) and inflammatory markers (TNFα and IL-6). Moreover, there was a significant increase in the levels of antioxidant enzymes (SOD, CAT, and GSH). Diosgenin appears to have the potential to protect testicular tissue from damage caused by the toxic effects of MTX through the reduction of oxidative stress and inflammation.