Enterovirus 71 (EV 71) is one of the commonest causative agents of hand, foot, and mouth disease (HFMD), which infects mainly young children. It has been associated with severe neurological complications worldwide, and has caused significant deaths in many provinces of China from March to May 2008. In this study, RNA interference (RNAi) was used as an antiviral agent to inhibit EV 71 replication in rhabdomyosarcoma (RD) cells. Three small interfering RNAs (siRNAs) targeting extremely conserved regions among multiple EV 71 strains in China could effectively block the replication of EV 71 strain Shzh-98. Combination transfection of these three siRNAs could produce a strong inhibitory effect not only in strain Shzh-98, but also in one epidemic strain Fuyang-0805 isolated from a child in the city of Fuyang with a clinical diagnosis of HFMD in 2008. These strategies and results suggest that RNAi has potential therapeutic use for the suppression of EV 71 infection in a broad spectrum of viral strains.