Fluoride, which is commonly found in drinking water and nutrients, threatens the environment and human life. Fluoride damages cells by causing free radical formation and affects male reproductive function by preventing spermatogenesis, changing the activity of spermatogenetic enzymes, and consequently leading to reduced reproductive capacity. A sufficient amount of antioxidants should be consumed in order to prevent oxidative stress of free radicals and minimize their effect. Vitamin C is a powerful antioxidant owing to its strong reductant activity. In this study, we treated TM4 Sertoli cells with different doses of fluoride (4 ppm and 20 ppm) for different time periods (24 hours and 48 hours) and additionally with vitamin C (50 ?M), and then analyzed the activity of enzymes involved in antioxidant and steroidogenic systems. According to our results, fluoride inhibits testosterone biosynthesis by reducing the activities of 3?-HSD and 17?-HSD enzymes, which have roles in testosterone synthesis in TM4 Sertoli cells, and vitamin C has a protective role against the damage of fluoride. Fluoride was found to cause oxidative damage in TM4 Sertoli cells by reducing the amount and activities of glutathione and antioxidant enzymes (SOD, CAT, GPx, GST, ?-GT), which function in the antioxidant system. A combination of fluoride and vitamin C was found to be protective against the damage by ROS and to cause an increase in the activities of enzymes in the intracellular antioxidant system. In conclusion, fluoride may result in harmful effects in TM4 Sertoli cells and vitamin C can be protective against these effects.