To investigate the in vitro anti-HIV-1effect of the aqueous extracts of Cordyceps sinensis. The aqueous stroma and sclerotium extracts were isolated from the fresh and dry Cordyceps sinensis specimen, respectively. The CCK-8test and the TZM-bl pseudovirus assay were used to examine the in-vitro cytotoxicity and anti-HIV-1activities of extracts. In addition, the reverse-transcriptase enzyme-activity assay and the surface plasma resonance(SPR)technology were taken to study the inhibition on the activity of reverse transcriptase and interaction with Vif protein. All 5aqueous extracts of Cordyceps sinensis exhibited in vitro anti-HIV-1effects,extracts from the fresh fungus showed more potent effect in inhibiting reverse-transcriptase activity than the dry fungus. Furthermore, a strong interaction was observed between the fresh stroma extract and Vif protein.The study clarified that the in vitro anti-HIV-1activity of the aqueous extracts of Cordyceps sinensis, may be mediated through inhibition of reverse-transcriptase activity and interaction with Vif protein.