Natural killer (NK) cells are critical in host innate defense against certain viruses. The role of NK cells in controlling hepatitis C virus (HCV) remains obscure. We examined whether NK cells are capable of inhibiting HCV expression in human hepatic cells. When NK cells are cultured with the HCV replicon-containing hepatic cells, they have no direct cytolytic effect but release soluble factor(s) suppressing HCV RNA expression. Media conditioned by NK cell lines (NK-92 and YTS) or primary NK cells isolated from healthy donors contain interferon γ (IFN-γ) and potently inhibit HCV RNA expression. Ligation of CD81 on NK cells inhibits IFN-γ production and results in decreased anti-HCV activity. In addition, the antibodies to IFN-γ or IFN-γ receptors abolish the anti-HCV activity of NK cell-conditioned media. The role of IFN-γ in NK cell-mediated, anti-HCV activity is supported by the observation that NK cell-conditioned media enhanced expression of signal transducer and activator of transcription-1, a nuclear factor that is essential in IFN-γ-mediated antiviral pathways. NK cell-conditioned media have the ability to stimulate intracellular IFN-α expression in the hepatic cells, suggesting a mechanism responsible for NK cell-mediated, anti-HCV activity. Thus, NK cells hold the potential to play a vital role in controlling HCV replication in hepatic cells using an IFN-γ-dependent mechanism.