The family of Picornaviridae includes six genera of positive, single-stranded RNA viruses.  The similarities of genomic organization in the genera of enteroviruses group of viruses are reflected in their similar replication strategies.  The parental viral RNA serves as a template for translation of a long polyprotein precursor, subsequently cleaved by three virus-encoded proteases, L^pro, 2A^pro and 3C^pro, to produce mature individual structural and nonstructural proteins.  These cleavages play a critical role in the viral life cycle.  Picornaviral proteases are cysteine proteases, but the overall protein folding for 2A^pro and 3C^pro resembles trypsin-type serine proteases.  Comparative studies of the three dimensional structures of these proteases have provided valuable insights into the understanding of substrate specificity and catalytic activity.  In addition to processing the viral polyprotein, picornaviral proteases also specifically cleave a number of host proteins and participate actively in the regulation of host cell survival and death.  Due to the unique protein structures and essential roles in viral replication and disease induction, these viral proteases have been viewed as rational targets for the development of antiviral drugs for the treatment of picornaviral infections.  In this article, we review recent advances in the study of functional roles of these proteases in viral pathogenesis, with particular focus on three human pathogenic genera, the Enterovirus, Rhinovirus and Heptatovirus.  We also summarize progress and future directions in the design and evaluation of picornaviral protease inhibitors.