To address the regulation and evolution of precursor protein cleavability in caliciviruses, we examined constraints on diversity of upstream regions of calicivirus precursor cleavage sites. We performed alanine scanning and supplementary mutagenesis of amino acids at P1, P2, P3, and P4 sites using four viruses representing the four major genera of the family Caliciviridae. This study complements previous mutagenesis studies and shows strong restrictions in mutations at the P1 and P4 sites for effective cleavage reactions. By contrast, such restrictions were less frequently observed at the P2 and P3 sites. Shannon entropy analysis of the reported sequences showed that the P2, P3, and P4 sites allow variations in amino acid size within a calicivirus genus whereas the P1 sites do not. Notably, the human sapovirus precursor protein exceptionally retains a basic rather than aromatic amino acid at the P4 site of the NS4/NS5 cleavage site in reported strains, and a substitution from basic to aromatic amino acid significantly enhanced cleavability at this site. Taken together, these data suggest the existence of (i) structural constraints on the P1 site that restrict size changes within each calicivirus genus, (ii) plastic substrate surfaces that accommodate size variation at the P2, P3, and P4 sites and modulate their own cleavabilities, and (iii) biological constraints on the P4 site that maintain the lower cleavability of the NS4/NS5 site in sapovirus.