Medicinal herbs are increasingly used in the search for safe and efficient drug candidates for hepatitis C virus infection. In this study, we have investigated the anti-HCV effect of compounds from Mori Cortex Radicis. During a screening for extracts with anti-HCV affinity from medicinal plants (173 species), the methanol extract of Mori Cortex Radicis was selected. Fractionation of the extract by monitoring antiviral activity with a replicon cell-based assay resulted in the isolation of five compounds, mulberroside C ( 1), moracin P ( 2), moracin O ( 3), moracin M ( 4) and mulberrofuran K ( 5) from the ethyl acetate-soluble fraction. Compounds 1 approximately 4 showed significant inhibitory activities. Compounds 2 and 3 showed potent inhibitory activity (IC (50) 35.6 microM, 80.8 microM, respectively) in the replicon cell assay, which was confirmed by NS3 helicase inhibitory activity (IC (50) 42.9 microM, 27.0 microM, respectively).