Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3′UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.