Introduction: Background and purpose: 5-Fluorouracil (5-FU) is among the most consumed anticancer drugs in the world, which, despite its widespread use in the treatment of various cancers, causes male fertility disorders. This study was conducted to investigate the antioxidant effect of folic acid on sperm parameters and oxidative stress in mice treated with 5-fluorouracil. Methods and Materials: In this experiment, 24 adult male NMRI mice (35-40 g) were divided into 4 groups (n=6) control, 5-fluorouracil (15mg/kg, 5 consecutive days), folic acid (1mg/kg, daily) and 5-fluorouracil + folic acid. Animals were treated intraperitoneally for 35 days. 24 hours after the last treatment, sperm samples were collected from the tail of the epididymis and used to evaluate the number, motility, viability, and daily sperm production. The level of testosterone hormone, malondialdehyde, and total serum antioxidant capacity were also measured. The data were statistically analyzed by One-Way ANOVA and Tukey's test and were considered significant at the level of (p<0.05). Results: A significant decrease in sperm parameters, testosterone hormone level, and total antioxidant capacity was observed in the 5-fluorouracil treatment group compared to the control group (p<0.05). The level of MDA in the 5-fluorouracil group showed a significant increase compared to the control group (p<0.05). In the simultaneous treatment group, folic acid reduced the toxic effects of 5-fluorouracil on the reproductive system of male mice and improved the above parameters. Conclusion: The results showed that folic acid as an antioxidant can induce its therapeutic and protective effect against the damage of the male reproductive system induced by fluorouracil, probably through inhibiting free radicals and oxidative stress and increasing the level of testosterone hormone.