Prenatal nicotine exposure (PNE) may lead to offspring's testicular dysplasia. Here, we confirmed the intergenerational effect of PNE on testosterone synthetic function and explored its epigenetic programming mechanism. Pregnant Wistar rats were injected subcutaneously with nicotine (2 mg/kg.d) from gestational day 9-20. Some dams were anesthetized to obtain fetal rats, the rest were allowed to spontaneous labor to generate F1 and F2 generation. In utero, PNE impaired testicular development and testosterone production. Meanwhile, the expression of steroidogenic acute regulatory protein (StAR) and 3β-hydroxysteroid dehydrogenase (3β-HSD) were decreased both in F1 and F2 generations. Furthermore, PNE enhanced the expression of fetal testicular nicotinic acetylcholine receptors (nAChRs) and histone deacetylase 4 (HDAC4), while obviously weakened histone 3 lysine 9 acetylation (H3K9ac) level of StAR/3β-HSD promoter from GD20 to postnatal week 12 and even in F2 generation. In vitro, nicotine increased nAChRs and HDAC4 expression, and decreased the StAR/3β-HSD H3K9ac level and expression, as well as the testosterone production in Leydig cells. Antagonism of nAChRs and inhibition of HDAC4 reversed the aforementioned changes. In conclusion, PNE programmed testicular low steroidogenesis and its heritability in male offspring rats. The underlying mechanism was associated to the low-level programming of StAR/3β-HSD H3K9ac via nAChR/HDAC4.