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‐γ). The investigation of the mechanisms for NK cell‐mediated anti‐HCV activity showed that NK SN‐treated hepatocytes expressed higher levels of IFN‐α/β 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.