BACKGROUND At present, the treatment of coxsackievirus-induced myocarditis remains difficult. Berberine (BBR), an isoquinoline alkaloid isolated from traditional medicine herbs, exhibits significant anti-viral efficacy against various viruses. However, the underlying mechanism by which BBR controls CVB3 infection has not yet been reported. The purpose of this study was to investigate the anti-viral efficacy of BBR against CVB3 infection and its mechanism. MATERIAL AND METHODS In our experiments, the protein levels of VP1 and MAPKs signal pathway were measured by Western blot. The mRNA level of VP1 was measured by RT-PCR. The virus titers were determined by TCID50 assay. RESULTS We found that BBR treatment significantly decreased CVB3 replication in HeLa cells. In addition, the BBR treatment reduced the phosphorylation levels of JNK and p38 MAPK upon CVB3 infection in both HeLa cells and primary rat myocardial cells. CONCLUSIONS Taken together, these results suggest that BBR inhibits CVB3 replication through the suppression of JNK and p38 MAPK activation, shedding new light on the investigation of therapeutic strategies against CVB3-induced viral myocarditis.