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The present study was designed to evaluate the protective effects of Reduning injection against Enterovirus 71(EV71) in Vero cells and in mice. The Vero cells were infected with 100 and 50 TCID50(50% tissue culture infective dose) of EV71, respectively. The inhibition of Reduning injection on cytopathic effect(CPE) was detected. Meanwhile, a mouse model produced by intraperitoneal EV71-infection(106 TCID50), was used to investigate the protective effects of Reduning injection. The total survival rate, living time, daily survival rate, weight ratio, and score for symptoms were examined. The viral loads in Vero cells and muscle tissues were detected using real-time PCR. Finally, the content of cytokines was analyzed by ELISA. In the Vero cells, 2.5 mg crude drug·m L-1 of Reduning injection inhibited CPE induced by EV71 infection. In the mice, 1.3 g crude drug·kg-1 of Reduning injection rescued death triggered by infection, in comparison with model group. Moreover, the survival rate, weight ratio, and clinical scores were also improved. The viral RNA copies in the Vero cells and the mice muscle tissues were reduced. Besides, the steep EV71-induced accumulations of TNF-α and MCP-1 were decreased by Reduning injection. In conclusion, Reduning injection showed promising protective effects against EV71 in Vero cells and in mice.