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Enterovirus infections are life-threatening viral infections which occur mainly among children and are possible causes of viral outbreak. Until now, treatment and management of infections caused by members of the genus Enterovirus largely depended on supportive care, and no antiviral medications are currently approved for the treatment of most of these infections. The urgency of discovering new therapeutic options for the treatment of enterovirus infection is increasing. In the present study, we identified that trans -2-hexenoic acid (THA), a natural product from a dietary source, possesses antiviral activity against coxsackievirus B (CVB) and enterovirus A71 (EV-A71) in a dose-dependent manner. We found that THA possesses antiviral activity at 50% effective concentrations (EC 50 ) of 2.9 μM and 3.21 μM against CVB3 and EV-A71 infections, respectively. The time of addition assay revealed that THA inhibits both CVB3 and EV-A71 replication at the entry stage of infection. Additional results from this study further suggest that THA inhibits viral replication by blocking viral entry. Given that THA has received approval as a food additive, treatment of enterovirus infections with THA might be a safe therapeutic option or could pave the way for semisynthetic manufacturing of more antiviral drugs in the future.