Enterovirus 71 (EV71) is the main causative agent of hand, foot, and mouth disease (HFMD) in young children. It is often associated with neurological complications and has caused high mortality levels in recent outbreaks in the Asia Pacific region. Currently, there is no effective antiviral therapy against EV71 infections. In this study, we have evaluated and compared the efficacies of three different forms of small interfering RNAs (siRNAs) in inhibiting EV71 replication in a murine model. We have shown that both synthetic 19-mer siRNAs and plasmid-borne short hairpin RNAs (shRNAs) targeted at the conserved 3D(pol) region were able to inhibit EV71 infections in suckling mice when delivered with or without lipid carrier via the systemic route. The treated mice did not exhibit hind limb paralysis and weight loss, as was observed in untreated mice. EV71 replication was significantly reduced as revealed by real-time reverse transcription polymerase chain reaction (RT-PCR) and Western blot. In addition, no evidence of interferon (IFN) induction was detected in the intestinal tissues harvested from the mice as a result of siRNA administration. However, the chemically synthesized 29-mer shRNA did not protect the suckling mice from EV71 infections despite being more potent in the in vitro system. Our results indicate that RNA interference (RNAi) may be a promising therapeutic approach for fighting EV71 infections.