In this study, we investigated the possible underlying mechanisms by which the UPS regulates CVB3 replication. We demonstrated that protein ubiquitination was enhanced after coxsackievirus infection. We further showed that knockdown of ubiquitin expression by small-interfering RNA (siRNA) decreased CVB3 infection, likely through the downregulation of ubiquitination and subsequent alteration of protein function and/or degradation. In addition, we showed that inhibition of deubiquitinating enzyme increased the inhibitory effects of proteasome inhibitors on CVB3 replication. We also found that CVB3 RNA-dependent RNA polymerase 3D (3D pol ) was modified by ubiquitination. Taken together, our study suggests an important role of ubiquitination in the regulation of coxsackieviral replication.