It is controversial whether exposure to isoflavones exerts male reproductive toxicity. The aim of this study was to investigate whether isoflavone exposure during adulthood could have deleterious impacts on male reproductive health by the cross-sectional study, animal experiments, and in vitro tests. In the cross-sectional study, we observed that urinary isoflavones were not significantly associated with semen quality including sperm concentrations, sperm count, progressive motility, and total motility, respectively. However, negative associations were found between plasma testosterone and urinary sigma isoflavones, genistein, glycitein, and dihydrodaidzein. In the animal experiments, serum and intratesticular testosterone levels were decreased in mice exposed to several dosages of genistein. Genistein administration caused upregulation of estrogen receptor alpha and downregulation of cytochrome P45017A1 protein levels in testes of mice. In vitro tests showed that genistein caused a concentration-dependent inhibition of testosterone production by TM3 Leydig cells. Elevated protein expression of estrogen receptor alpha and decreased messenger RNA/protein level of cytochrome P45017A1 were also observed in genistein-treated cells. Protein level of cytochrome P45017A1 and testosterone concentration were significantly restored in the estrogen receptor alpha small interferring RNA-transfected cells, compared to cells that treated with genistein alone. The results demonstrate that exposure to isoflavones during adulthood may be associated with alterations of reproductive hormones. Particularly, genistein, which inhibits testosterone biosynthesis through upregulation of estrogen receptor alpha in Leydig cells of mice, might induce the disruption of testosterone production in human. The present study provides novel perspective into potential targets for male reproductive compromise induced by isoflavone exposure. Isoflavone exposure during adulthood is remarkably associated with reproductive hormone perturbation in males, which may be mediated through the estrogen receptor alpha/cytochrome P45017A1 pathway.