Hepatitis C virus (HCV) infection is a severe liver disease that often leads to liver cirrhosis and hepatocellular carcinoma (HCC). Current therapy is inadequate to conquer this viral disease. In this study, we identified parthenolide (1), an active component in feverfew, a popular remedy for fever and migraine, as a lead compound with an EC50 value of 2.21 microM against HCV replication in a subgenomic RNA replicon assay system. Parthenolide is able to potentiate the interferon alpha-exerted anti-HCV effect. Several commercially available sesquiterpene lactones (2-5) structurally analogous to parthenolide and a series of synthesized Michael-type adducts of parthenolide (12-18) also exhibit micromolar concentrations for anti-HCV activities. Structure-activity relationship was elucidated to reveal that the spatial arrangement of the terpenoid skeleton fused with an alpha-methylene-gamma-lactone moiety produces maximal anti-HCV activity. In addition, a strong anti-HCV potency indicates a possibility of secondary amino adducts (12-18) converting back to parthenolide or being replaced by the nucleophilic residues of proteins inside cells. This work shows that screening of natural products is a viable and fast way for identifying novel molecular diversity as potential drug leads.