In the present study, we constructed several siRNA-expressing plasmids targeted to conserved sequences within the coding regions of viral polymerase protein 3D, capsid protein VP4 and nonstructural (NP) protein 2B of the FMDV genome. Some of the plasmids inhibited several isolates of serotype O and serotype Asia I FMDV in baby hamster kidney (BHK-21) cells and suckling mice. Furthermore, we reported that siRNA directed against the polymerase protein 3D of FMDV and delivered by attenuated S. choleraesuis were capable of inhibiting virus replication in guinea pigs and swine.