Infection with hepatitis C virus (HCV) is one of the major global health problems, approximately 170 million people are infected worldwide. The chronic HCV infection is associated with a high risk of developing liver cirrhosis, hepatocellular carcinoma (HCC) and liver failure. Unfortunately, there is still no effective vaccine or antibodies available for the prevention of infection. RNA interference (RNAi) represents a promising new approach to combat viral infections, and recent developments in the field of gene therapy have increased the feasibility of clinical applications. RNAi techniques have made rapid progress in the basic understanding of HCV biology and revealed numerous new viral and host-cell factors as potential targets for therapy. Together with the improvement of gene delivery technique and the discovery of the critical role of microRNA (miRNA) in HCV infection, RNAi and miRNA-based antiviral strategies hold great promise for the future. In this article, we provide a comprehensive overview of current developments of therapeutic targets of RNAi, liver-targeted delivery systems and the potential applications of miRNAs in treatment of hepatitis C infection.