Hepatitis B virus(HBV) infection is a severe health problem in the world. However,there is still not a satisfactory therapeutic strategy for the HBV infection. To search for new anti-HBV agents with higher efficacy and less side-effects,the inhibitory activities of traditional Chinese medicine Rheum palmatum L. ethanol extract(RPE) against HBV replication were investigated in this study. Quantitative real-time polymerase chain reaction(PCR) was employed to analyze the inhibitory activity of RPE against HBV-DNA replication in a stable HBV-producing cell line HepAD38; the expression levels of HBV surface antigen(HBsAg) and e antigen(HBeAg) were also determined by enzyme linked immunosorbent assay(ELISA) after RPE treatment. RPE could dose-dependently inhibit the production of HBV-DNA and HBsAg. The concentration of 50% inhibition(IC50) was calculated at 209.63,252.53μg /mL,respectively. However,its inhibitory activity against HBeAg expression was slight even at high concentrations. RPE had a weak cytotoxic effect on HepAD38 cells(CC50= 1 640μg /mL) and the selectivity index(SI) was calculated at 7.82. Compared with two anthraquinone derivatives emodin and rhein,RPE showed higher ability of anti-HBV and weaker cytotoxicity. So Rheum palmatum L. might possess other functional agents which could effectively inhibit HBV-DNA replication and HBsAg expression. Further purification of the active agents,identification and modification of their structures to improve the efficacy and decrease the cytotoxicity are required.