Host heat shock cognate 70 (Hsc70) protein is packaged into hepatitis C viral (HCV) particles as a structural component of the virus in the assembly process. It helps HCV RNA release into the cytoplasm in the next infection cycle. The goal of this study is to investigate whether chemically down-regulating host Hsc70 expression could be a novel strategy to interrupt HCV replication. Compounds were screened with an Hsc70 messenger RNA (mRNA) assay. IMB-DM122 was found to be an effective and safe inhibitor for Hsc70 mRNA/protein expression in human hepatocytes. IMB-DM122 inhibited HCV replication through destabilization of Hsc70 mRNA, and the half-life of host Hsc70 mRNA was reduced by 78% after the compound treatment. The Hsc70 mRNA 3′ untranslated region sequence is the element responsible for the effect of IMB-DM122 on Hsc70 mRNA. The compound appears to be highly efficient in inhibiting Hsc70-related HCV replication. Treatment of the HCV-infected hepatocytes with IMB-DM122 reduced the virion encapsidation of Hsc70, and therefore disrupted HCV replication and the infection cycle. IMB-DM122 showed considerable good safety in vitro as well as in vivo with no indication of harmful effect on liver and kidney functions. Conclusion: Hsc70 might be a new drug target and mechanism to inhibit HCV proliferation. (HEPATOLOGY 2010;)