Astragalus polysaccharide (APS) is the most immunoreactive substance in Astragalus. APS can regulate the body's immunity and is widely used in many immune related diseases. However, till now, there is little information about its contribution to the protection of astrocytes infected by virus. Toll-like receptor 3 (TLR3) is a key component of the innate immune system and has the ability to detect virus infection and trigger host defence responses. This study was undertaken to elucidate the protective effect of APS on herpes simplex virus (HSV-1) infected astrocytes and the underlying mechanisms. The results showed that APS protected the astrocytes from HSV-1 induced proliferation inhibition along with increasing expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) markedly. Moreover, APS significantly promoted the expression of Toll-like receptor 3 (TLR3) and the activation of nuclear factor-κB (NF-κB) in astrocytes. In addition, while astrocytes were pretreated with TLR3 antibody before adding HSV-1 and APS, the expression of TLR3, TNF-α, and IL-6 and the activation of NF-κB decreased sharply. These results indicate that APS can protect astrocytes by promoting immunological function provoked by HSV-1 through TLR3/NF-κB pathway.