Enterovirus A71 (EV71) infection can cause hand-foot-and-mouth disease and severe neurological complications in children. However, the biological processes regulated by EV71 remain poorly understood. Herein, proteomics and metabonomics studies were conducted to uncover the mechanism of EV71 infection in Rhabdomyosarcoma (RD). RD cells and identify potential drug targets. Differential expressed proteins from enriched membrane were analyzed by isobaric Tags for Relative and Absolute Quantitation (iTRAQ)-based proteomics technology. Twenty-six differential proteins with 1.5-fold (p < 0.05) change were detected, including 14 up-regulated proteins and 12 down-regulated proteins. The up-regulated proteins are mainly involved in metabolic process, especially in the glycolysis pathway. Of which, Aalpha-enolase protein was found to increase with temporal dependence following EV71 infection. The targeted metabolomics analysis revealed that glucose absorption and glycolysis metabolites were increased after EV71 infection. Then theThe glycolysis pathway was inhibited by knocking down alpha-enolase (ENO1) or the use of a glycolysis inhibitor (dichloroacetic acid, DCA); and we found that EV71 infection were inhibited by depleting ENO1 or using DCA. Totally, our Our study indicates that EV71 may reprogram glucose metabolism by activating glycolysis, and EV71 infection can be inhibited by interrupting the glycolysis pathway. ENO1 and DCA may be a potential targets against EV71; and DCA could act as an inhibitor of or inhibitors against EV71.