The role of natural killer (NK) cells in controlling hepatitis C virus (HCV) infection and replication has not been fully delineated. We examined NK cell-mediated noncytolytic effect on full cycle HCV infection of human hepatocytes. Human hepatocytes (Huh7.5.1 cells) co-cultured with NK cells or treated with supernatants (SN) from NK cells cultures had significantly lower levels of HCV RNA and protein than control cells. This NK cell-mediated anti-HCV activity could be largely abolished by antibody to interferon-gamma (IFN-gamma). The investigation of the mechanisms for NK cell-mediated anti-HCV activity showed that NK SN-treated hepatocytes expressed higher levels of IFN-alpha/beta than the control cells. NK SN also enhanced IFN regulatory factor-3 and 7 expression in the hepatocytes. In addition, NK SN enhanced the expression of signal transducer and activator of transcription 1 and 2, the nuclear factors that are essential for the activation of IFN-mediated antiviral pathways. These data provide direct evidence at cellular and molecular levels that NK cells have a key role in suppressing HCV infection of and replication in human hepatocytes.