Particulate Matter 2.5 (PM_2.5) disrupts endocrine functions and may negatively affect sperm quality and quantity in males; however, the long-term effects and potential mechanisms of this effect are unknown. This study aimed to investigate the epigenetic mechanism of maternal exposure to PM_2.5-induced inhibin B hypermethylation in male offspring. In this experiment design, pregnant C57BL/6 mice were treated with two doses of PM_2.5 (4.8 and 43.2 mg/kg bw). The membrane control group was given a sampling membrane and the control group received nothing. Following the formation of the vaginal plug, intratracheal instillation of PM_2.5 was administered every three days until delivery of the pups. To assess the effect of PM_2.5 in vitro, TM4 cells, a Sertoli-like cell line, was treated with different concentrations (0, 25, 50, 100 μg/mL) of PM_2.5 for 24 h. The results displayed that Sperm motility, as well as the number of adult offspring, was decreased in the PM_2.5 exposed group relative to the untreated controls. Increased vacuolization was observed in the Sertoli cells of mice that were exposed to PM_2.5 in utero. The levels of inhibin and testosterone were reduced and the levels of LH and FSH increased in the PM_2.5 groups relative to the untreated controls. In vitro, PM_2.5 resulted in cell cycle inhibition as well as increased apoptosis in TM4 cells. Moreover, PM_2.5-induced inhibin B hypermethylation and activation of the p21/Cleaved Caspase-3 pathway resulted in TM4 cell apoptosis that was rescued through the use of a DNA methylation inhibitor. Together, our data suggest that prenatal exposure to PM_2.5 results in inhibin B hypermethylation and can activate the p21/Cleaved Caspase-3 pathway, resulting in Sertoli cell apoptosis, aberrant secretion of androgen binding protein, and decreased testosterone, thus resulting in the inhibition of spermatogenesis.