Hepatitis C virus (HCV) interferes with interferon (IFN)-mediated innate immune defenses. Toll-like receptor (TLR) 7 agonists robustly inhibit HCV infection. We hypothesize that HCV infection may interfere with the expression and/or function of TLR7, a sensor of single-stranded RNA. We identified reduced TLR7 RNA and protein levels in hepatoma cells expressing HCV (full-length, BB7-subgenomic, and JFH-1 clone) compared with control HCV-naive cells. The biological relevance of this finding was confirmed by the observation of decreased TLR7 RNA in livers of HCV-infected patients compared with controls. HCV clearance, by IFN-alpha treatment or restrictive culture conditions, restored the decreased TLR7 expression. Treatment with RNA polymerase inhibitors revealed a shorter TLR7 half-life in HCV-replicating cells compared with controls. Downstream of TLR7, an increased baseline IRF7 nuclear translocation was observed in HCV-positive cells compared with controls. Stimulation with the TLR7 ligand R837 resulted in significant IRF7 nuclear translocation in control cells. In contrast, HCV-replicating cells showed attenuated TLR7 ligand-induced IRF7 activation. CONCLUSION: Reduced TLR7 expression, due to RNA instability, directly correlates with HCV replication and alters TLR7-induced IRF7-mediated cell activation. These results suggest a role for TLR7 in HCV-mediated evasion of host immune surveillance.