Encephalomyocarditis virus (EMCV) is recognized as a pathogen inducing acute myocarditis and sudden death in preweaned piglets and severe reproductive failure in sows. In this study, eight specific small interfering RNA (siRNA) duplexes targeting different genomic regions of EMCV BJC3 were designed and their ability to inhibit virus replication in BHK-21 cells was investigated. The results showed that BHK-21 cells transfected with siRNA duplexes to 2C gene (JH-4,666, BJC-1,739), 2B gene (BJC-807), 3C gene (BJC-2,363) and 3D gene (BJC-3269) were specifically resistant to EMCV infection when exposed to 500 times the 50% cell culture infective dose (CCID(50)) of EMCV. The levels of the 3D gene in the transfected cells were obviously decreased. IFA and Western blotting analysis confirmed that the expression of VP1 protein in cell culture transfected with the siRNAs was apparently reduced. Of the five siRNAs, JH-4,666, BJC-2,363 and BJC-3,269 were the most effective. Combination of the siRNA duplexes enhanced the inhibition of EMCV replication. Our data indicated that specific siRNAs are able to inhibit the replication of porcine encephalomyocarditis virus in BHK-21 cells, suggesting that RNAi might provide a new approach to prevent EMCV infection.