There is a paucity of studies on the multigenerational reproductive toxicity of fine particle matter (PM2.5) exposure during pregnancy on male offspring and the underlying mechanisms. This study explored the effects of PM2.5 exposure during pregnancy on the spermatogenesis of three consecutive generations of male mouse offspring. We randomized pregnant C57BL/6 mice into the control group, the Quartz Fiber Membrane control group, and two experimental groups exposed to different concentrations of PM2.5 (4.8 and 43.2 mg/kg B.Wt.). Pregnant mice from experimental groups received intratracheal instillation of PM2.5 of different doses on a three-day basis until birth. F1 mature male offspring from PM2.5-exposed pregnant mice were mated with normal female C57BL/6 mice. Likewise, their F2 mature male followed the same to produce the F3 generation. The results showed that PM2.5 exposure during pregnancy led to decreased body and tail length, body weight, and survival rates, decreased sperm concentration and sperm motility, and increased sperm abnormality rates significantly in F1 male offspring. We barely observed significant impacts of PM2.5 on the birth number, survival rates, and index of testes in the F2 and F3 offspring. Further exploration showed that PM2.5 exposure during pregnancy caused the morphological abnormality of Sertoli cells, downregulated androgen receptor (AR) and connexin43, upregulated anti-Müllerian hormone (AMH), cytokeratin-18 (CK-18), caspase-3, and cleaved caspase-3, decreased thyroid-stimulating hormone (TSH) and testosterone (T), and increased triiodothyronine (T3) in F1 male mouse offspring. Overall, we hypothesize that PM2.5 exposure during pregnancy mainly negatively impacts spermatogenesis in the F1 offspring. The possible mechanism could be that PM2.5 exposure during pregnancy disrupts endocrine hormone release in the F1 generation, thereby influencing the maturation and proliferation of their Sertoli cells and hindering spermatogenesis. This study for the first time investigates the role of Sertoli cells in the reproductive toxicity of PM2.5 on offspring.