NK cells are important innate immune effector cells, normally characterized as CD56+ CD3− lymphocytes. In this study, we report that CD56− CD16+ NK cells expand in many patients with chronic hepatitis C virus infection. These CD56− NK cells were functionally impaired with respect to cytokine production upon target cell recognition, in comparison to CD56 dim and CD56 bright NK cell subsets. In particular, CD56− NK cells were strikingly defective in their polyfunctional response as measured by the coexpression of MIP-1β, IFN-γ, TNF-α, and CD107a degranulation. The ability of these cells to mediate three or four of these functions was poor; expression of MIP-1β alone dominated their response. CD56− NK cells retained expression of receptors such as the natural cytotoxicity receptors and NKG2D, whereas the expression of CD57 and perforin was lower when compared with CD56 dim NK cells. Interestingly, pretreatment levels of CD56− NK cells correlated with the outcome of pegylated IFN-α and ribavirin treatment. In patients with CD56− NK cells in the range of healthy subjects, 80% reached a sustained virological response to treatment, whereas only 25% of patients with levels clearly above those in healthy subjects experienced a sustained virological response. Thus, chronic hepatitis C virus infection is associated with an expansion of CD56− NK cells functionally skewed toward MIP-1β production only. Furthermore, high levels of these cells reveal a disturbance in innate cellular immunity that is associated with an impaired ability to respond to antiviral treatment with IFN-α and ribavirin.