Enteroviruses (EVs) are single-stranded RNA viruses with positive polarity whose members cause widespread human diseases with major health and economic impacts, commonly observed in young children and adults. They are the most common etiologic agents of viral respiratory and neurological infections, usually causing mild symptoms but capable of causing severe pathology, including acute asthma exacerbations, acute flaccid paralysis, and myocarditis. There is currently no approved antiviral treatment, in part because of the great phenotypic diversity of enteroviruses. The molecular mechanisms by which enteroviruses cause these diseases are still poorly understood, but increasing evidence points to two enteroviral proteases, 2Apro and 3Cpro, as key players in pathogenesis. 2Apro performs posttranslational proteolytic processing of viral polyproteins and cleaves several host proteins to evade the antiviral immune response and promote viral replication. Therefore, studies of 2Apro could reveal additional substrates that may be associated with specific diseases.