Objective: Coxsackievirus 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 a cysteine protease, 3C protease (3Cpro). Herein, we report the development of new, water-soluble, anti-Coxsackieviral (CVB) 3Cpro inhibitor (3CproI) by CVB3 myocarditis murine model. Methods and Results: Since human rhinovirus (HRV) 3CP and CVB 3CP are similar in their catalytic structures, we had reported an insoluble peptidomimetic inhibitor of CVB 3Cpro, which has naphthyl group. We substituted the naphthyl group with various hetero-aromatic groups. Antiviral activity of CVB 3CproI derivatives in vitro were evaluated by the measurement of cytotoxic effect in HeLa cells. We found that isoxazole ring of 4-yl quinoline inhibitor showed the most potent antiviral activity. In vivo, we infected 4-week-old Balb/c male mice by intraperitoneal inoculation of 104 PFU of CVB3 (n=25 with 3CproI, n=35 without 3CPI) at day 0. And we injected new water soluble 3CproI (500M/100l) once a day, intraperitoneally from day 2 to day 14. The 3-week-survival rates of 3CPI-treated mice were significantly higher than those of control mice (72%, 20% on each group, P < 0.01 among groups). Myocardial inflammation and virus titers were all significantly reduced in the 3CproI treated groups compared to the controls. Conclusion: Herein, we successfully develop new water soluble 3CproI, protein structure-based drug which inhibited activity of 3Cpro and had no direct cytopathic effect in high dose. Consequently, 3CPI inhibited the proliferation of CVB3. In vivo, CVB 3CPI significantly attenuated the CVB3-induced inflammation, mortality and virus replication. It could be used as a novel therapeutic agent for CVB3-induced myocarditis.