Objective To explore the reproductive toxicity of 2,4-D butylate to the testis in male mice.Methods Forty-eight ICR male mice were randomly divided into four groups: the control group, and three 2,4-D butylate experimental groups (10, 20, 40 mg/kg), 12 mice in each group. 2,4-D butylate was intragastrically administered once a day and six days per week for five weeks. At the end of the exposure, the activities of total antioxidant capacity (T-AOC), Na⁺ K⁺ -ATPase, Ca⁺⁺ Mg⁺⁺-ATPase, lactate dehydrogenase (LDH) and succinate dehydrogenase (SDH) in testis homogenate were measured by spectrophotometry. Results The activity of T-AOC was gradually decreased with the increase of doses, with a significant difference between the high dose group and other groups. The activities of LDH in the moderate and high dose groups were significantly lower than those of the low dose group and control group, and there was a significant difference between the high dose group and moderate dose group. The activities of SDH in the testis was gradually decreased with the increase of the 2,4-D butylate dose, showing significant differences between the high dose group and the moderate dose and control groups, and between the high and moderate dose groups and the low dose group. The activities of Na⁺K⁺-ATPase in the moderate and high dose groups were significantly lower than that of the control and low dose group. The activities of Ca⁺⁺Mg⁺⁺-ATPase was significantly lower in the experimental groups than that in the control group. Conclusion Exposure to 2,4-D butylate has certain toxic effect on the testicular tissue in male mice.