Ribavirin is used as a component of combination therapies for the treatment of chronic hepatitis C virus (HCV) infection together with pegylated interferon and/or direct-acting antiviral drugs. Its mechanism of action, however, is not clear. Direct antiviral activity and immunomodulatory functions have been implicated. Plasmacytoid dendritic cells (pDCs) are the principal source of type 1 interferon during viral infection. The interaction of pDCs with HCV-infected hepatocytes is the subject of intense recent investigation, but the effect of ribavirin on pDC activation has not been evaluated. In this study we showed that ribavirin augments toll-like receptors 7 and 9-mediated IFNα/β expression from pDCs and up-regulated numerous interferon-stimulated genes. Using the H77S.3 HCV infection and replication system, we showed that ribavirin enhanced the ability of activated pDCs to inhibit HCV replication, correlated with elevated induction of IFNα. Our findings provide novel evidence that ribavirin contributes to HCV inhibition by augmenting pDCs-derived type 1 IFN production.