Small interfering RNAs (siRNAs) efficiently inhibit gene expression by RNA interference. Here, we report efficient inhibition, by both synthetic and vector‐derived siRNAs, of hepatitis C virus (HCV) replication, as well as viral protein synthesis, using an HCV replicon system. The siRNAs were designed to target the 5′ untranslated region (5′ UTR) of the HCV genome, which has an internal ribosomal entry site for the translation of the entire viral polyprotein. Moreover, the 5′ UTR is the most conserved region in the HCV genome, making it an ideal target for siRNAs. Importantly, we have identified an effective site in the 5′ UTR at which ∼80% suppression of HCV replication was achieved with concentrations of siRNA as low as 2.5 nM. Furthermore, DNA‐based vectors expressing siRNA against HCV were also effective, which might allow the efficient delivery of RNAi into hepatocytes in vivo using viral vectors. Our results support the feasibility of using siRNA‐based gene therapy to inhibit HCV replication, which may prove to be valuable in the treatment of hepatitis C.