This work investigated the effects of Allium ampeloprasum extract (AME) on testosterone synthesis in TM3 Leydig cells under hydrogen peroxide (H2O2)- induced oxidative stress. AME showed no increment of cell proliferation in the oxidatively stressed cells. Testosterone level lowered when the cells were treated with H2O2, but increased in the presence of AME. To determine the effect of AME on the testosterone biosynthetic pathway, we examined the expression level of the proteins involved in the synthesis or degradation of testosterone. The protein expression levels of CYP11A1, 3β-HSD2 and 17β-HSD3 (which are involved in testosterone synthesis) were decreased by H2O2 but increased by the AME treatment. Furthermore, the protein expression levels of 5α-reductase2 and aromatase (which are involved in testosterone degradation) were increased by H2O2 but decreased by the AME treatment. In conclusion, AME increased the testosterone level by increasing the expression of testosterone synthetase and decreasing the expression of the testosterone converting enzyme in TM3 Leydig cells under H2O2-induced oxidative stress. These results indicated that AME can alleviate andropause syndrome by restoring testosterone.