Highly active antiretroviral therapy (HAART) has been successful in reducing HIV-1-associated morbidity and mortality since its introduction in 1996. It, however, fails to eradicate HIV-1 infection thoroughly. The high cost of life-long HAART and the emergence of drug resistance among HIV-1-infected individuals have brought renewed pressure for the discovery of novel antivirals and alternative medicines. Traditional Chinese medicine (TCM) is one of the mainstreams of complementary and alternative medicine, and serves as rich resources for new drug development. Despite almost 100 plant-derived compounds are in clinical trials, few target HIV-1 infection. In this study, we discovered that extract of Sanguisorba officinalis (SOE) has anti-HIV-1 activities. Using a cell-based assay and single-cycle luciferase reporter viruses pseudotyped with envelopes from HIV-1 or control viruses, we found that SOE exhibited significant inhibitory ability against both CCR5 and CXCR4 tropic HIV-1 (ADA and HXB2) with respective IC50 values of 1.91±0.16 μg/ml and 3.70±0.53 μg/ml. Interestingly, SOE also inhibited SIV infection but failed to block vesicular stomatitis virus (VSV), SARS-CoV and influeunza H5N1 pseudoviruses. Furthermore, we showed that SOE had no effects on post-entry events of HIV-1 replication. It blocked entry by acting on viral envelope directly because SOE pre-treatment with the virus but not with cell lines expressing viral receptors showed the maximal inhibitory activity. In addition, SOE was able to inhibit reverse-transcription-inhibitor-resistant viruses (K103N, Y188L, and K103N/Y188L/G190A) and a protease-inhibitor-resistant strain (PI-2840). Our findings demonstrated SOE as a novel and specific entry inhibitor, which shed lights on the discovery of anti-HIV-1 drugs from traditional herbal medicines.