Background: Methotrexate, a chemotherapy agent used in cancer treatment, increases reactive oxygen species (ROS) and oxidative stress. Rutin, a flavonoid with potent antioxidant and anti-inflammatory effects, can counteract oxidative damage by neutralizing free radicals. This study examined rutin’s protec- tive role on biochemical markers in the testicular tissue and se- rum of NMRI male mice exposed to methotrexate. Materials and Methods: In this study, twenty-four adult NMRI male mice (average weight: 31 ± 3 g) were randomly divided into four groups: control, methotrexate (20 mg/kg/bw), rutin (100 mg/kg/bw), and methotrexate + rutin, and they were treated for 35 days. Following dissection, the serum samples were collected to measure the serum levels of total antioxidant capacity (TAC) and malondialdehyde (MDA). Additionally, testicular tissue samples were used to measure the levels of TAC and MDA. Data were statistically analyzed using SPSS software version 26, with One-Way ANOVA and Tukey's test. The mean difference was considered significantly different at P<0.05. Results: The methotrexate group showed a significant reduction in serum and testicular tissue TAC level when compared to the control group (P<0.01). Serum and testicular tissue MDA lev- els increased significantly in the methotrexate group compared to the control group (P<0.01). However, in the methotrexate + rutin group, serum and testicular tissue TAC level showed a sig- nificant increase compared to the methotrexate group (P<0.05). Furthermore, a significant decrease in the mean serum and tis- sue MDA level was observed in the methotrexate + rutin group when compared to the methotrexate group (P<0.01). Conclusion: Our results showed that Rutin reduces methotrex- ate-induced oxidative stress in NMRI male mice by improving TAC and reducing MDA levels in both serum and testicular tis- sue. These findings suggest that rutin may serve as a potential antioxidant agent to mitigate methotrexate-induced oxidative damage in reproductive tissue.
