RNA interference (RNAi) is a sequence-specific, post-transcriptional process of mRNA degradation induced by small interfering RNA molecules. In this report, RNAi strategy was exploited to treat the infection of enterovirus 71 (EV71), considered as one of the most virulent pathogens that can cause severe complications in the family of Picornaviridae. We developed short hairpin RNA (shRNA) expression plasmids that significantly inhibited viral protein expression in a sequence-specific and dose-dependent fashion after transient transfection in cell cultures. Stable expression of shRNAs in cultured cells exhibited marked viral resistance in every step assessed in the viral replication. Using cytotoxicity of shRNA-expressing cells as a surrogate marker, it was shown that replication of EV71 was specifically attenuated by these plasmid-derived shRNAs, while replications of other related enteroviruses examined were not. These proof-of-concept studies demonstrated the feasibility of this approach for the therapy of EV71-associated diseases.