Purpose <p>Perfluorooctane sulfonic acid (PFOS) is a representative persistent organic pollutant that has been shown to impair male reproductive function. Epidemiological studies and animal models suggest that vitamin D may modify male reproductive function. However, whether 1α,25(OH)₂D₃ could mitigate the PFOS-induced damage of Sertoli cells, and the underlying mechanisms remain poorly defined.</p> Methods <p>Mouse Sertoli cell line TM4 were pretreated with or without 10 nM or 100 nM 1α,25(OH)₂D₃ for 6&#xa0;h, followed by 100 µM or 200 µM PFOS for 24&#xa0;h. Cell viability was assessed using the CCK8 assay. Reactive oxygen species (ROS) were detected by fluorescent staining, while malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH) levels were measured using commercial kits. siRNA transfection, quantitative real-time PCR (qRT‒PCR), and Western blotting were performed to elucidate the underlying mechanisms. Molecular docking studies were conducted to explore the potential binding of PFOS to Keap1.</p> Results <p>PFOS increased MDA contents, decreased GSH levels, and induced cytotoxicity in mouse Sertoli cells. Mechanistically, PFOS might directly bind to Keap1, facilitating its degradation, leading to persistent activation of the Nrf2-ARE pathway. Conversely, 1α,25(OH)₂D₃ attenuated PFOS-induced cytotoxicity by alleviating the Nrf2-ARE pathway, a protective effect which was abolished by vitamin D receptor (VDR) knockdown.</p> Conclusion <p>PFOS induces cytotoxicity in Sertoli cells by binding to Keap1 and persistently activating the Nrf2-ARE pathway. 1α,25(OH)₂D₃ emerges as a promising candidate for mitigating PFOS-induced testicular dysfunction.</p> Graphical abstract <p></p>