To explore the antiviral effect and mechanism of polysaccharide from Spirulina platensis(PSP) on herpes simplex virus type 2 (HSV-2), a standard strain of HSV-2 (333 strain) was used to investigate the antiviral effect of PSP in vitro. PSP in various concentrations was applied to different stages of HSV-2 replication cycle. Finally, the virus infectivity (TCID50), cytopathic effect (CPE),and MTT staining method for viable cells (MTT assay) were used as markers to evaluate the effect of PSP on HSV-2. The quantity of HSV-DNA was detected by real-time fluorescence quantitative PCR(FQ-PCR). The HSV-2 infected Vero cell ultrastructures were observed by transmission electron microscopy (TEM). The results showed that PSP had little cytotoxic effect on Vero cells, it could not directly inactivate HSV-2 infectivity. PSP not only interfered in adsorption of HSV-2 to Vero cells but also inhibited HSV-2 biosynthesis in the cells. FQ-PCR results showed that the inhibitory rate on HSVDNA also increased in a dose-dependent and time-dependent manner. TEM also confirmed that PSP exhibited pronounced inhibitory effect on HSV-2. In conclusion, the antiviral effect of PSP on HSV-2 may be attributed to the inhibition of virus adsorption, virus replication and synthesis in cells.