Picornavirus family members cause disease in humans. Human rhinoviruses (RV), the main causative agents of the common cold, increase the severity of asthma and COPD; hence, effective agents against RVs are required. The 2A proteinase (2A(pro)), found in all enteroviruses, represents an attractive target; inactivating mutations in poliovirus 2A(pro) result in an extension of the VP1 protein preventing infectious virion assembly. Variations in sequence and substrate specificity on eIF4G isoforms between RV 2A(pro) of genetic groups A and B hinder 2A(pro) as drug targets. Here, we demonstrate that although RV-A2 and RV-B4 2A(pro) cleave the substrate GAB1 at different sites, the 2A(pro) from both groups cleave equally efficiently an artificial site containing P1 methionine. We determined the RV-A2 2A(pro) structure complexed with zVAM.fmk, containing P1 methionine. Analysis of this first 2A(pro)-inhibitor complex reveals a conserved hydrophobic P4 pocket among enteroviral 2A(pro) as a potential target for broad-spectrum anti-enteroviral inhibitors.