The Picornaviridae family comprises a diverse group of small RNA viruses that cause a variety of human and animal diseases. Some of these viruses are known to induce cleavage of components of the innate immune system and to inhibit steps in the interferon pathway that lead to the production of type I interferon. There has been no study of the effect of picornaviral infection on the events that occur after interferons have been produced. To determine whether members of the Enterovirus genus can antagonize the antiviral activity of interferon-stimulated genes (ISGs), we pretreated cells with alpha interferon (IFN-alpha) and then infected the cells with poliovirus type 1, 2, or 3; enterovirus type 70; or human rhinovirus type 16. We found that these viruses were able to replicate in IFN-alpha-pretreated cells but that replication of vesicular stomatitis virus, a Rhabdovirus, and encephalomyocarditis virus (EMCV), a picornavirus of the Cardiovirus genus, was completely inhibited. Although EMCV is sensitive to IFN-alpha, coinfection of cells with poliovirus and EMCV leads to EMCV replication in IFN-alpha-pretreated cells. The enteroviral 2A proteinase (2A(pro)) is essential for replication in cells pretreated with interferon, because amino acid changes in this protein render poliovirus sensitive to IFN-alpha. The addition of the poliovirus 2A(pro) gene to the EMCV genome allowed EMCV to replicate in IFN-alpha-pretreated cells. These results support an inhibitory role for 2A(pro) in the most downstream event in interferon signaling, the antiviral activities of ISGs.