Background/Aims: While hepatic stellate cells (HSC) are known to be key mediators of liver fibrosis, only little is known about their functional role in the innate immune system of the liver. Methods: To address this question, murine HSC were isolated from livers of C57BL/6J mice and human HSC were isolated from liver samples obtained from resections and liver explants. HSC were stimulated with Toll-like receptor (TLR) 1-9 ligands for 20 h. Supernatants were harvested and used in virus protection assays (encephalomyocarditis virus, EMCV) as well as in human and murine hepatitis C virus (HCV) replicon systems. Expression of interferon (IFN), retinoic acid-inducible gene-I (RIG-I), and interferon-stimulated genes (ISGs) was assessed by quantitative reverse transcription polymerase chain reaction. Results: While all TLRs were detectable in HSC, in murine HSC only TLR 3 and -4 agonists could induce cytokines that had an antiviral effect upon EMCV and HCV replication. IFN-β was the main cytokine mediating the antiviral activity of TLR 3-stimulated HSC whereas other cytokines of undefined nature were involved in TLR 4-mediated antiviral effects. In human HSC, only TLR 3 stimulation led to production of antiviral cytokines. The antiviral effect was related to the up-regulation of ISGs and RIG-I in target cells. Conclusions: These data demonstrate that murine and human HSC have as yet unrecognized antiviral properties when activated through the TLR-system and TLR 3/HCV in particular. This sheds new light on their role in the innate immune system of the liver and their participation in the control of HCV replication. © 2009 European Association for the Study of the Liver.