Bisphenol AF (BPAF) is a newly identified contaminant in the environment that has been linked to impairment of the male reproductive system. However, only a few studies have systematically studied the mechanisms underlying BPAF-induced toxicity in testicular Sertoli cells. Hence, this study primarily aims to explore the toxic mechanism of BPAF on the porcine Sertoli cell line (ST cells). The effects of various concentrations of BPAF on ST cell viability and cytotoxicity were evaluated using the Counting Kit-8 (CCK-8) assay. The results demonstrated that exposure to a high concentration of BPAF (above 50 μM) significantly inhibited ST cell viability due to marked cytotoxicity. Flow cytometry analysis further confirmed that BPAF facilitated apoptosis and induced cell cycle arrest in the G2/M phase. Moreover, BPAF exposure upregulated the expression of pro-apoptotic markers BAD and BAX while downregulating anti-apoptotic and cell proliferation markers BCL-2, PCNA, CDK2, and CDK4. BPAF exposure also resulted in elevated intracellular levels of reactive oxygen species (ROS) and malondialdehyde (MDA), alongside reduced activities of the antioxidants glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD). Furthermore, the ROS scavenger N-acetyl-L-cysteine (NAC) effectively blocked BPAF-triggered apoptosis and cell cycle arrest. Therefore, this study suggests that BPAF induces apoptosis and cell cycle arrest in ST cells by activating ROS-mediated pathways. These findings enhance our understanding of BPAF’s role in male reproductive toxicity and provide a foundation for future toxicological assessments.