Abstract: Objective To investigate the anti-human immunodeficiency virus-1 (HIV-1) activity of Euonymus fortunei extract and its mechanism, providing a theoretical basis for the research and development of traditional Chinese herbal resources in the treatment of acquired immunodeficiency syndrome (AIDS). Methods The peripheral blood mononuclear cells (PBMCs)-HIV-1bal cell model system was used as the experimental model, and the effect of Euonymus fortunei extract on the cell activity and anti-HIV-1 activity of PBMCs was investigated by CellTiter-Glo assay and HIV-1 p24 antigen assay. Additionally, the mechanism of anti-HIV-1 action of Euonymus fortunei extract was determined through the time-of-addition assay and HIV-1 enzymatic assays. Results In the PBMCs-HIV-1bal cell model, Euonymus fortunei extract showed no significant effect on cellular activity at the highest concentration, with a median cytotoxic concentration (CC50)>40 μg/mL. Within the non-cytotoxic concentration range, the level of HIV-1bal decreased with the increase of the extract concentration, with the IC50=0.084 μg/mL, and the SI>476.19. Time-of-addition assay revealed that Euonymus fortunei extract had the highest inhibitory activity on HIV-1IIIB when it was added to cells at the same time as the virus, and the inhibitory rate of HIV-1 activity decreased at 2 h and gradually lost its inhibitory effect on the virus at 18-24 h, similar to the known HIV entry inhibitor Enfuvirtid (T-20). Key HIV-1 enzyme activity experiments found that the extract had no significant inhibitory effect on reverse transcriptase activity but exhibited dose-dependent inhibition of integrase activity, with integrase activity decreasing with increasing extract concentration (IC50=0.18 μg/mL); The extract could inhibit the activity of HIV-1 protease in a certain concentration range (IC50=39.18 μg/mL). Conclusions In this study, it was found that Euonymus fortunei extract has a strong inhibitory activity against HIV-1 activity, which may exert anti-HIV-1 effects by acting on viral entry into cells and inhibiting integrase activity, and has a good application prospect in the research and development of multi-target new drugs against HIV-1.