Total ethanol extract and saponins from Chinese herb radix Astragali (huangqi) have been previously shown to possess anti-hepatitis B virus (HBV) activities in vitro. To identify the active ingredients, we isolated a triterpenoid saponin that was determined to be astragaloside IV. In the human HBV-transfected liver cell line HepG(2) 2.2.15, astragaloside IV effectively suppressed secretion of HBV antigens with inhibition rates of 23.6% for the secretion of Hepatitis B surface antigen (HBsAg) and 22.9% for that of Hepatitis B e antigen (HBeAg) at 100 microg/ml after 9 d of treatment. The inhibitory activity of astragaloside IV on secretion of HBV antigens is more potent than that of 3TC without significant cytotoxicity. In duck hepatitis B virus (DHBV)-infected ducklings, astragaloside IV caused 64.0% inhibition at 120 mg/kg, 49.6% inhibition at 40 mg/kg, and 41.7% inhibition at 10 mg/kg to serum DHBVs after 10 d of treatment, and also reduced serum DHBV DNA levels. Together, our results demonstrate that astragaloside IV possesses potent anti-HBV activity.