Interferon-alpha (IFN-alpha) is the most commonly used therapeutics for the treatment of chronic viral infection. However, many viruses are resistant to IFN-alpha treatment to some degrees through encoding inhibitors of the IFN-alpha producing or signaling pathway. Multiple HCV viral proteins have been reported to be involved in IFN-alpha resistance. To develop a method to screen for factors that inhibit the antiviral activity of IFN-alpha, a mini-library of HCV genome was transduced into the Huh7 cells containing the HCV subgenomic replicon (CON1 HCV S2204I) and screened for the factor that rendered the cells more resistant to IFN-alpha treatment. A fragment of nonstructural protein 4B (NS4B), named tNS4B, was isolated. Expression of tNS4B or the full-length NS4B in CON1 HCV S2204I or naïve Huh7 cells inhibited the protection of the cells by IFN-alpha treatment from vesicular stomatitis virus (VSV) infection. In Huh7 cells expressing NS4B or tNS4B, IFN-alpha-induced phosphorylation levels of signal transducer and activator of transcription 1 (STAT1) were reduced. Furthermore, expression of NS4B reduced IFN-alpha-induced expression levels of type I interferon receptor and a reporter driven by the ISRE promoter. In conclusion, we have developed a method to screen for IFN-alpha resistance factors and identified HCV NS4B as such a factor.