Chronic infection with hepatotropic viruses is the main cause of chronic liver disease and cirrhosis worldwide. Toll-like receptor 3 (TLR3) and Toll-like receptor 7 (TLR7) are pathogen-recognition receptors that are expressed on innate immune cells. They recognize viral RNA, which induces their activation, with a subsequent increase in type I interferon transcription. Hepatitis C virus (HCV) infection inhibits the expression of TLR3 and TLR7; however, the mechanism by which this occurs is unclear. MicroRNAs (miRNAs) are small RNAs that posttranscriptionally regulate gene expression. Their aberrant expression is commonly correlated with disease status, as is the case with HCV infection. Here, we found that miR-758 levels were increased in patients with HCV infection and were correlated with TLR3 and TLR7 expression levels in the patients with HCV infection, and bioinformatics analysis predicted that TLR3 and TLR7 are targets of miR-758. Therefore, we postulate that HCV may increase the level of miR-758, which inhibits the expression of TLR3 and TLR7, resulting in a loss of antiviral effect. In order to test our hypothesis, we constructed an HCV core protein expression plasmid and used it to transfect liver cells. The results showed that HCV infection increased miR-758 levels and decreased TLR3/TLR7 expression. Furthermore, using RT-PCR and luciferase reporter analysis, we found that miR-758 targets TLR3 and TLR7, with a subsequent decrease in IFNα and IFNβ production. In conclusion, our results highlight the upregulation of miR-758 expression by HCV as a novel mechanism contributing to downregulation of TLR3 and TLR7 in patients with HCV infection.