Coxsackieviruses are important human pathogens that induce myocarditis and pancreatitis. However, there are no vaccines or therapeutic reagents for their clinical treatment. Although RNA interference (RNAi)-based approaches to the prevention of viral production have been developed recently, limitations to the in vivo delivery systems and variations in the viral target sequences still hamper the strategy. In this study, to overcome these limitations, we have constructed recombinant lentivirus-delivered short hairpin RNAs (shRNAs) against sequences in highly conserved cis-acting replication element (CRE) within the 2C protein of coxsackievirus B3 (CVB3), designated MET-2C. A recombinant lentivirus, designated Met-2C lenti, was constructed that contains the MET-2C sequence, which acts as a shRNA. Met-2C lenti clearly reduced viral production in CVB3-infected cells in vitro. Moreover, the mice injected intraperitoneally with Met-2C lenti had significant reductions in viral titers, viral myocarditis, and proinflammatory cytokines after challenge with CVB3, compared with those in GFP lenti infected control mice. Moreover, Met-2C lenti improved survival rate compared with that of the GFP lenti infected control group. Therefore, Met-2C lenti is potentially a clinical therapeutic agent for the treatment of viral myocarditis.