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HCV (hepatitis C virus) research, including therapeutics and vaccine development, has been hampered by the lack of suitable tissue culture models. Development of cell culture systems for the growth of the most drug-resistant HCV genotype (1b) as well as natural isolates has remained a challenge. Transfection of cultured cells with adenovirus-associated RNAI (VA RNAI), a known interferon (IFN) antagonist and inhibitor of dsRNA-mediated antiviral pathways, enhanced the growth of plasma-derived HCV genotype 1b. Furthermore, persistent viral growth was achieved after passaging through IFN-α/β-deficient VeroE6 cells for 2 years. Persistently infected cells were maintained in culture for an additional 4 years, and the virus rescued from these cells induced strong cytopathic effect (CPE). Using a CPE-based assay, we measured inhibition of viral production by anti-HCV specific inhibitors, including 2′-C-Methyl-D-Adenosine, demonstrating its utility for the evaluation of HCV antivirals. This virus constitutes a novel tool for the study of one of the most relevant strains of HCV, genotype 1b, which will now be available for HCV life cycle research and useful for the development of new therapeutics., Author Summary Hepatitis C virus (HCV) causes a persistent infection that can lead to hepatocellular carcinoma and liver cirrhosis. Interferon (IFN)-based treatments are ineffective for some HCV genotypes. HCV research has been hampered by the lack of suitable cell culture systems. With the discovery of a unique HCV genotype 2a isolate that can replicate in the human liver cell line Huh7, some obstacles were overcome. However, there remains the need of systems to grow IFN-resistant genotypes and serum-derived isolates. Here we show that the presence of adenovirus-associated RNAI (VA RNAI), a known IFN antagonist, permitted establishment of a persistent infection of genotype 1b in VeroE6 cells that were passaged weekly for more than 2 years. The persistent virus induces strong cytopathic effect (CPE), a feature that allowed the development of a CPE-based assay to test HCV-specific inhibitors, neutralization by anti-HCV immunoglobulins and by anti-CD81 antibody, and HCV-specific siRNA. Our system provides the first persistent culture of genotype 1b virus and a convenient assay that can be used for therapeutics development.