Phytosterols (PS) are plant origin sterols naturally found in many foods and added as food additives. Since 1950, PS have attracted considerable attention due to their ability to lower serum cholesterol and inhibit cardiovascular disease (CVD). However, recent studies have found that PS act as endocrine-disrupting chemicals in laboratory animals. Therefore, this study was aimed at finding the mechanism(s) for PS effects on the sexual behaviors and reproductive functions in male Japanese quails (Coturnix coturnix japonica). At 15 d of age, 30 male Japanese quails were randomly assigned to 3 groups for the chronic in-vivo experiment. Animals were gavaged daily with single dose of PS suspension (PS dissolved in medium chain triglyceride [MCT]) into the crop sac from 15-100 d of age. Following maturation, a sexual behavior test, semen collection, and test of fertilization ability were performed. Blood was collected by cervical dislocation at 100 day of age for hormones analysis. To observe the direct effects of PS on the testis, interstitial cells of the normal testes were cultured for 24 h. Ovine-LH (O-LH) was used for half of each group to stimulate interstitial cells for testosterone production. The results showed that chronic doses of PS reduced (P < 0.01) mount and copulation behavior in male quails (primarily in the group receiving 800 mg/kg BW). Both acute in vitro and chronic in vivo experiments revealed a reduction in testosterone (P < 0.05) after PS treatments; concomitantly, Leydig cell numbers were also low (P < 0.05) at a dose of 800 mg/kg BW. Real-time PCR results showed lower expression of 17β-hydroxysteroid dehydrogenase (17β-HSD) at the same dose. The results suggested that feeding large amounts of PS reduced testosterone levels and sexual behavior by affecting Leydig cell proliferation, and cholesterol trafficking, 17β-HSD expression in the testes of male Japanese quail.