Infection with hepatitis C virus (HCV) is a leading cause of chronic liver disease worldwide. Little is known about how this virus is able to persist or whether this persistence might be because of its ability to alter the early innate immune response. The major HCV envelope protein E2 has been shown to bind to CD81. Thus, HCV binding to natural killer (NK) cells could result in the cross-linking of CD81. To explore this possibility, we investigated whether cross-linking CD81 on NK cells could alter NK cell function. CD81 cross-linking by monoclonal antibody (mAb) specific for CD81 or by immobilized E2 have been shown to result in costimulatory signals for human T cells. In this study, we show that CD81 cross-linking via immobilized E2 or mAbs specific for CD81 inhibits not only non major histocompatibility complex-restricted cytotoxicity mediated by NK cells but also interferon (IFN)-gamma production by NK cells after exposure to interleukin (IL)-2, IL-12, IL-15, or CD16 cross-linking. These results show that CD81 cross-linking mediates completely different signals in NK cells versus T cells. Importantly, these results suggest that one mechanism whereby HCV can alter host defenses and innate immunity is via the early inhibition of IFN-gamma production by NK cells.