Enteroviruses are major causative agents of various human diseases, and some of them are currently considered to be an enormous threat to public health. However, no effective therapy is currently available for the treatment of these infections. We identified gemcitabine, a nucleoside-analog drug used for cancer treatment, from a screen of bioactive chemicals as a novel inhibitor of coxsackievirus B3 (CVB3) and enterovirus 71 (EV71). Gemcitabine potently inhibited the proliferation of CVB3 and EV71, as well as the replication of CVB3 and EV71 replicons, in cells with a low micromolar IC50 (1-5 muM). Its strong inhibitory effect was also observed in cells infected with human rhinoviruses, demonstrating broad-spectrum antiviral effects on enteroviruses. Mechanistically, an extensive analysis excluded the involvement of 2C, 3A, IRES-dependent translation, and also that of polyprotein processing in the antiviral effects of gemcitabine. Importantly, gemcitabine in combination with ribavirin, an antiviral drug currently being used against a few RNA viruses, exhibited a synergistic antiviral effect on the replication of CVB3 and EV71 replicons. Consequently, our results clearly demonstrate a new indication for gemcitabine as an effective broad-spectrum inhibitor of enteroviruses and strongly suggest a new therapeutic strategy using gemcitabine alone or in combination with ribavirin for the treatment of various diseases associated with enterovirus infection.