Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located within the 5′-nontranslated region (5′NTR). We investigated the effect of interferon alfa (IFN-α) on the IRES-directed translation of HCV, using two stably transformed cell lines, RCF-1 and RCF-26, of Huh7 cells derived from human hepatocellular carcinoma that express dicistronic reporter proteins, Renilla luciferase (RL) and firefly luciferase (FL), separated by HCV-IRES. After the administration of IFN-α or poly(I)-poly(C), HCV-IRES–directed translation was inhibited in a dose-dependent manner. The relative HCV-IRES activity (F/L) decreased to 60% at 5,000 IU/mL of IFN-α and 45% at 40 μg/mL of poly(I)-poly(C). Thus, IFN-α or poly(I)-poly(C) inhibited HCV-IRES–directed translation more efficiently than a cellular cap–dependent translation. 2′,5′–oligoadenylate synthetase (2′,5′AS) protein level in cells analyzed significantly increased after the administration of IFN-α, but not upon poly(I)-poly(C). Overexpression of double-stranded RNA-activated protein kinase (PKR) gene did not mimic the selective inhibition of HCV-IRES–directed translation in the transformant cells, suggesting that neither the 2′,5′AS nor the PKR system are involved in this selective inhibition. Interestingly, the expression of the autoantigen, La, which has been reported to enhance HCV-IRES–directed translation, was significantly reduced after the administration of IFN-α and poly(I)-poly(C) in a dose-dependent manner. Transient expression of La protein completely restored the selective inhibition of HCV-IRES–directed translation by IFN-α and poly(I)-poly(C). These findings suggested a new antiviral mechanism induced by IFN-α in that IFN-α or poly(I)-poly(C) selectively inhibited HCV-IRES–directed translation compared with the eukaryotic cap-dependent translation through the reduction of La protein.