Objective: To investigate the effect of sodium polyanethol sulfonate (SPS) on herpes simplex virus type 1 (HSV-1) infection in vitro. Human corneal epithelial (HCE-T) cells and Vero cells were infected with HSV-1 [HSV-1 f strain, HSV-1f; HSV-1-H129 with green fluorescent protein (GFP) knock-in, HSV-1g]. SPS was added to the culture medium at various concentrations in time-of-addition assay. Experiments including photography of fluorescence in HSV-1g or plaque formation by HSV-1f, western blot assays, real-time RT-PCR assays, cytopathic effect inhibition assays, cytotoxicity assays, and viral absorption and penetration assays were performed to explore the antiviral effect and mechanism of the compounds. We identified that SPS reduced the replication of HSV-1 in HCE-T and Vero cells in a dose-dependent manner. HSV-1g fluorescence was reduced by 66.3% and 65.4% in HCE-T and Vero cells, respectively, after treatment with 0.4 µg/ml SPS. Furthermore, the viral fluorescence intensities were inhibited by SPS in a dose-dependent manner when the viruses or cells were preincubated with SPS. Relative levels of the ICP4 protein and VP16 mRNA were decreased by SPS in a dose-dependent manner. Moreover, the IC50 values of SPS for HSV-1g and HSV-1f in HCE-T cells were 0.69±0.09 μg/ml and 1.63±0.44 μg/ml, respectively. Even 10,000 µg/ml SPS had no obvious cytotoxicity toward HCE-T and Vero cells. Importantly, viral absorption and penetration assays showed that the relative fluorescence intensity of HSV-1g was significantly reduced by SPS in a dose-dependent manner in the absorption test, but no change was observed in the penetration test. SPS inhibits HSV-1 replication in HCE-T and Vero cells, indicating that SPS has the potential for treating HSV-1 infection, particularly HSV-1 keratitis. Copyright © 2022 Molecular Vision.