Coxsackievirus B3 (CVB3) is one of the most prevalent pathogens of viral myocarditis, which may persist chronically and progress to dilated cardiomyopathy. We have previously demonstrated a critical role of the ubiquitin/proteasome system (UPS) in the regulation of coxsackievirus replication in mouse cardiomyocytes. In the present study, we extend our interest to an in vivo animal model to examine the regulation and role of the UPS in CVB3-induced murine myocarditis. Male myocarditis-susceptible A/J mice at age 4-5 weeks were randomized to four groups: sham + vehicle (n=10), sham + proteasome inhibitor (n=10), virus + vehicle (n=20) and virus + proteasome inhibitor (n=20). Proteasome inhibitor was administered subcutaneously once a day for 3 days. Mice were sacrificed on day 9 post-infection and infected heart was harvested for Western blot, plaque assay, immunostaining, and histological examination. We showed that CVB3 infection led to an accumulation of ubiquitin conjugates at 9 day post-infection. Protein levels of ubiquitin-activating enzyme E1A/E1B, ubiquitin-conjugating enzyme UBCH7, as well as deubiquitinating enzyme UCHL1 were markedly increased in CVB3-infected mice as compared to sham infection. However, there was no significant alteration in proteasome activities at 9 day post-infection. Immunohistochemical staining revealed that increased expression of E1A/E1B was mainly localized to virus damaged cells. Finally, we showed that application of a proteasome inhibitor significantly reduced CVB3-induced myocardial damage. This observation reveals a novel mechanism of coxsackieviral pathogenesis, and suggests that the UPS may be an attractive therapeutic target against coxsackievirus-induced myocarditis.