Epidemiologic studiessuggested the association between prenatal Di-(2-ethylhexyl) phthalate (DEHP) exposure and disorders of sex development (DSD), adult male disorders, and reproductive aging. However, the evidence is still weak, and the underlying mechanism hasn’t been revealed. Mono-(2-ethylhexyl) phthalate (MEHP) is the main bioactive metabolite of DEHP. Inhibiting testosterone synthesis by interfering with steroidogenic gene expression induces testicular toxicity. So prenatal DEHP exposure may induce lifelong testicular toxicity by continuously interfering with steroidogenic gene expression. In this study, male mice underwent different doses (0, 100, 500, 1000mg/kg) of prenatal DEHP exposure, the testicular toxicity (genital development, testosterone, semen quality, and morphology of testis tissue) in the neonatal, post-puberal and middle-aged stages was observed, and the steroidogenic gene (Lhcgr, Star, Cyp11a1, Cyp17a1, Hsd17b3, and Hsd3b2) expression was analyzed by qPCR and WB. We also explored the interference of steroidogenic gene expression in TM3 Leydig cells after MEHP exposure. As a result, prenatal DEHP exposure induced lifelong testicular toxicity including instant testicular injury, DSD, and reproductive aging. The male mice with prenatal exposure manifested as poor genital development and reduced testosterone synthesis, poor semen quality, and phylogeneticseminiferous tubules, especially in the high dose (1000mg/kg). Prenatal DEHP exposure continuously interfered with steroidogenic gene expression. MEHP reduced testosterone synthesis of TM3 Leydig cells by interfering with steroidogenic gene expression. In conclusion, prenatal DEHP exposure induces lifelong testicular toxicity by continuously interfering with steroidogenic gene expression, thus indicating the association between prenatal exposure and DSD, adult male disorders, and reproductive aging. Environmental Implication DEHP, a widely applied plasticizer, is easily contacted by pregnant women and causes prenatal exposure of male offspring. Epidemiologic and animal studies indicate that prenatal DEHP exposure is associated with male genital malformation, as well as adult male disorders (infertility, low testosterone). Our study explores the lifelong testicular toxicity in male mice and the key role of interfering with steroidogenic gene expression on testicular toxicity following prenatal DEHP exposure. As a result, prenatal DEHP exposure induced lifelong testicular toxicity including instant testicular injury, DSD, and reproductive aging by continuously interfering with steroidogenic gene expression.