Objective To investigate the toxic effect and molecular mechanism of silicon dioxide nanoparticles (SiO₂NPs) on mouse Sertoli cells (TM4), TM4 cells were exposed to medium containing various concentrations of SiO₂NPs (0, 1, 10, 100 mg/L) for 24 h.Methods After treatment, the morphology and viability of TM4 cells were detected by optical microscope and cell counting kit (CCK-8). The level of intracellular reactive oxygen species (ROS) was measured by fluorescent probe DCFH-DA, and the content of malondialdehyde (MDA) and activity of superoxide dismutase (SOD) were detected by kits according to the manufacturers’ protocol. The percentage of apoptotic cells in TM4 cells was detected by Annexin V-FITC/PI kit, and the expression levels of apoptotic molecules including Fas, FasL, Caspase-8, Caspase-3, Bax and Bcl-2 were detected by Western blot.Results The results showed that SiO₂NPs inhibited the cell survival rates, decreased cell concentrations and changed cell morphology of TM4 cells in a dose-dependent manner. In addition, the level of ROS in TM4 cells was significantly up-regulated after exposure, followed by an increase in the content of MDA and the activity of SOD. Further study found that SiO₂NPs increased cell apoptosis rate and activated the pro-apoptotic signaling pathway mediated by Fas/FasL. Interestingly, inhibition of oxidative stress by NAC in TM4 cells can alleviate the cell injure and apoptosis induced by SiO₂NPs.Conclusion In summary, SiO₂NPs induce TM4 cells apoptosis through oxidative stress and activation of Fas/FasL signaling pathway.