Previous studies have demonstrated that the polycomb repressor Bmi1 is universally expressed in all types of testicular cells and might regulate the spermatogonia proliferation, however, it is unclear whether Bmi1 plays a critical role in maintaining normal male fertility in vivo. To answer this question, we first confirmed that Bmi1 is universally expressed in all types of testicular cells and found that the gene relative expression levels of Bmi1 in testis were the highest relative to other organs. Then we investigated the role of Bmi1 in maintaining normal male fertility using Bmi1 knockout male mouse model. Our results demonstrated that Bmi1 deficiency resulted in totally male infertility with smaller testis, severe oligospermia and sperm malformation. Mechanistically, decreased serum testosterone levels with down-regulating 3βHSD and 17βHSD expression levels, reduced germ cell proliferation, increased germ cell apoptosis with up-regulating p16, p19, p53 and p21 expression levels, increased reactive oxygen species (ROS) and H2O2 levels with down-regulating gene expression levels of anti-oxidant enzymes, and increased 8-OHdG and γ.H2AX positive cells in testis were observed in Bmi1 deficient mice compared with wild-type mice. These results indicate that Bmi1 deficiency results in male infertility by reducing testosterone syntheses, increasing oxidative stress and DNA damage, activating p16 and p19 signaling pathway, inhibiting germ cell proliferation and inducing germ cell apoptosis and sperm malformation. Thus, Bmi1 may be a novel and potential target for the clinic treatment of male infertility.