Bisphenol AF (BPAF), one of the most common bisphenol analogues, has been reported to exhibit higher estrogenic activity compared to bisphenol A (BPA) due to the presence of additional hydrophobic groups. To comprehensively understand the male reproductive toxicity of BPAF, TM3 Leydig cells were used to investigate the effects of BPAF on cell proliferation, apoptosis, and cell cycle arrest. The underlying mechanisms of cellular responses induced by BPAF were examined through analysis of target mRNA and protein expression. Results showed that BPAF treatment reduced cell viability and induced both G2/M cell cycle arrest and apoptosis in a time- and dose-dependent manner in TM3 Leydig cells. RNA sequencing analysis and experimental verification further revealed that the p53 signaling pathway was involved in BPAF-induced cytotoxicity. Furthermore, Pifithrin-α (PFT-α), a p53 inhibitor, attenuated BPAF-induced G2/M cell cycle arrest and apoptosis. These results demonstrate that the p53 signaling pathway mediates BPAF-induced cell cycle arrest and apoptosis in Leydig cells, providing mechanistric insights into BPAF's toxicological effects on the male reproductive system.