Enteroviruses dramatically remodel the cellular infrastructure for efficient replication and curtailing host antiviral responses. The roles of viral proteins in these processes have been studied mostly in vitro, by ectopic overexpression, or by surrogate infection systems, all of which have shortcomings. Here, we replace the essential 2A cleavage site at the P1-P2 junction with an internal ribosome entry site (IRES), 3CD cleavage site, or T2A sequence, allowing us to catalytically inactivate 2Apro in the virus context. Viruses with an inactive 2A^pro are hampered in replication in cell lines and are severely attenuated in a Coxsackievirus B3 (CVB3) mouse pancreatitis infection model. We show that 2A^pro is essential for disturbing nucleocytoplasmic transport, shutting down host mRNA translation, suppressing stress granule formation, suppressing the induction of the IFN response, and overcoming IFN-induced restriction factors. Moreover, using an advanced single-molecule live cell imaging approach, we reveal that 2A^pro is important for the initial round of replication of the incoming viral RNA, which is a bottleneck for efficient infection. Thus, 2A^pro plays a critical role in subverting antiviral responses and establishing a favorable environment to expedite enterovirus replication.