Chronic hepatitis C virus (HCV) infection is a leading cause of cirrhosis, liver failure, and hepatocellular carcinoma. Although the combination therapy employing pegylated interferon (IFN)-α and ribavirin is effective, this treatment is effective in only approximately 50% patients with genotype 1 HCV infection. IFN-γ is a potent anti-HCV agent that exhibits its antiviral action through a receptor distinct from that for IFN-α. Therefore, IFN-γ application might provide an alternative approach to IFN-α-based therapies. However, recombinant IFN-γ protein exhibits a poor pharmacokinetic property, that is, a very short half-life. It is our hypothesis that sustained IFN-γ serum concentrations produced by gene transfer could effectively eliminate HCV in vivo. We examined the in vivo antiviral activity in human hepatocyte chimeric mice infected with genotype 1b HCV at high HCV RNA titers (10(5)-10(7) copies/ml). The human IFN-γ-expressing plasmid vector pCpG-huIFNγ exhibited prolonged transgene expression in mice compared with the plasmid vector pCMV-huIFNγ. Moreover, the gene transfer of pCpG-huIFNγ eliminated HCV from the liver of the chimeric mice for a sustained period. On the contrary, administration of pCMV-huIFNγ could not eliminate HCV. In conclusion, we found that a single pCpG-huIFNγ injection resulted in long-term elimination of HCV RNA in chimeric mice, providing, for the first time, direct evidence that chronic infection with high titer HCV in vivo can be treated by sustained IFN-γ treatment.