Objective: Coxsackievirus B3 is a primary cause of viral myocarditis. The viral genome encodes a single polyprotein that undergoes a series of proteolytic events to produce several viral proteins. Most of this proteolytic processing is catalyzed by the 3C protease, which is a cysteine protease that cleaves the viral polyprotein encoded by the viral genome. We investigated the potential for chemotherapeutic use of CVB 3CPI. Methods and Results: Since human rhinovirus (HRV) 3CP and CVB 3CP are similar in their catalytic structures, we have synthesized derivatives of an existing peptidomimetic inhibitor of HRV 3CP. We substituted the ethyl group at the P2��� location of the HRV 3CP inhibitor with hydrophobic aromatic rings, that can interact preferentially with the Tyr residue. The resulting derivatives showed dramatically increased inhibitory activities against CVB 3CP. Antiviral activity of CVB 3CPI derivative in vitro was evaluated by measurement of cell cytotoxic effect in dose dependent manner. In GFP expression, 3CPI inhibited the proliferation of GFP-H3, which is replication competent virus. GFP expression decreased in high dose of 3CPI. Also we observed that 3CPI inhibited cleavage of viral capsid protein (VP2/VP4) and viral proliferation by western blotting assay. Conclusion: 3CPI, protein structure-based drug, inhibited activity of 3C protease and were not observed cytopathic effect in high dose. Consequently, 3CPI inhibited the proliferation of CVB3. It could be used as a novel therapeutic agent for CVB3-induced myocarditis.