BACKGROUND: Fine particulate matter [particulate matter (PM) with aerodynamic diameter of  ≤ 2.5 μm (PM2.5)] is considered a major component of ambient PM. Exposure to PM2.5 was shown to be associated with male reproductive system injury. Ferroptosis is regarded as an iron-dependent programmed cell death that is associated with the pathological process. It has been reported that SIRT1 has protective effects on the male reproductive system. However, the underlying mechanisms of exposure to PM2.5-induced testicular injury are still unexplored. OBJECTIVES: In this study, we investigated the relationship between ferroptosis and male reproductive injury after exposure to PM2.5 and the role of SIRT1/HIF-1α signaling pathway in this process. METHODS: We established a PM2.5 exposure model in vivo and in vitro using Sertoli cell Sirt1 conditional knockout C57BL/6 (cKO) mice testes and primary Sertoli cells. Hematoxylin and eosin (H&E) staining were conducted to examine the histology of the mice testes. Sperm parameters and biotin tracer assay were conducted to evaluate the effects of exposure to PM2.5 on the mice testes. Related markers and genes related to the blood-testis barrier (BTB) and ferroptosis were measured by quantitative real-time polymerase chain reaction (qPCR), western blot, and immunofluorescence assay. siRNA transfection was used to evaluate the potential mechanism. RESULTS: Significant pathological damage and lower sperm quality were detected in mice testes exposed to PM2.5. We found that exposure to PM2.5 damaged the BTB and inhibited the expression level of the BTB-related proteins (including Connexin 43, Occludin, Claudin 11, N-Cadherin and ZO-1). According to the enrichment analysis results, ferroptosis and HIF-1α signaling pathway were significantly enriched in mice testes and primary Sertoli cells exposed to PM2.5. Subsequent experiments were conducted to verify the results of the enrichment analysis and revealed differences in the expression levels of HIF-1α, ferroptosis-related genes (including GPX4, SLC7A11, ACSL4, and HO-1) and ferroptosis-related markers [including malondialdehyde (MDA), glutathione (GSH), and Fe2+], associated with lower expression of SIRT1 after exposure to PM2.5. These results suggest that PM2.5 exposure may be associated with ferroptosis and HIF-1α signaling pathway in male reproductive dysfunction. CONCLUSIONS: Taken together, in vivo and in vitro experiments verified that PM2.5 exposure in mice may lead to testicular dysfunction through new pathways. https://doi.org/10.1289/EHP14447.