Antisense oligodeoxynucleotides (AS-ODNs) are promising therapeutic agents for the treatment of virus-induced diseases. We previously reported that coxsackievirus B3 (CVB3) infectivity could be inhibited effectively in HeLa cells by phosphorothioate AS-ODNs complementary to different regions of the 5' and 3' untranslated regions of CVB3 RNA. The most effective target is the proximal terminus of the 3' untranslated region. To further investigate the potential antiviral role of the AS-ODN targeting this site in cardiomyocytes (HL-1 cell line), corresponding AS-ODN (AS-7) was transfected into the HL-1 cells and followed by CVB3 infection. Analyses by RT-PCR, Western blotting and plaque assay demonstrated that AS-7 strongly inhibits viral RNA and viral protein synthesis as compared to scrambled AS-ODNs. The percent inhibitions of viral RNA transcription and capsid protein VP1 synthesis were 87.6 and 40.1, respectively. Moreover, AS-7 could inhibit ongoing CVB3 infection when it was given after virus infection. The antiviral activity was further evaluated in a CVB3 myocarditis mouse model. Adolescent A/J mice were intravenously administrated with AS-7 or scrambled AS-ODNs prior to and after CVB3 infection. Following a 4-day therapy, the myocardium CVB3 RNA replication decreased by 68% and the viral titers decreased by 0.5 log(10) in the AS-7-treated group as compared to the group treated with the scrambled AS-ODNs as determined by RT-PCR, in situ hybridization and viral plaque assay. Taken together, our results demonstrated a great potential for AS-7 to be further developed into an effective treatment towards viral myocarditis as well as other diseases caused by CVB3 infection.