The 2A proteinase (2A(pro)) is an enterovirally encoded cysteine protease that plays essential roles in both the processing of viral precursor polyprotein and the hijacking of host cell translation and other processes in the virus life cycle. Crystallographic studies of 2A(pro) from enterovirus 71 (EV71) and its interaction with the substrate are reported here. EV71 2A(pro) was comprised of an N-terminal domain of a four-stranded antiparallel beta sheet and a C-terminal domain of a six-stranded antiparallel beta barrel with a tightly bound zinc atom. Unlike in other 2A(pro) structures, there is an open cleft across the surface of the protein in an open conformation. As demonstrated by the crystallographic studies and modeling of the complex structure, the open cleft could be fitted with the substrate. On comparison 2A(pro) of EV71 to those of the human rhinovirus 2 and coxsackievirus B4, the open conformation could be closed with a hinge motion in the bII2 and cII beta strands. This was supported by molecular dynamic simulation. The structural variation among different 2A(pro) structures indicates a conformational flexibility in the substrate-binding cleft. The open structure provides an accessible framework for the design and development of therapeutics against the viral target.