The molecular determinants of substrate specificity and selectivity in the proprotein convertase (PC) family of proteases are poorly understood. Here we demonstrate that the natural serpin family inhibitor, serpin B8, is a specific and selective inhibitor of furin relative to the other PCs of the constitutive protein secretion pathway, PC4, PC5, PACE4, and PC7 (PC4-PC7, respectively), and identify reactive-site (P6-P5' residues) and exosite elements of the serpin that contribute to this specificity and selectivity through studies of chimeras of serpin B8 and α1PDX, an engineered serpin inhibitor of furin. Kinetic studies revealed that the specificity and selectivity of the serpin chimeras for inhibiting PCs were determined by P6-P5 and P3-P2 residue-dependent recognition of the P4Arg-X-X-P1Arg PC consensus sequence and exosite-dependent recognition of the reactive loop P2' residue of the chimeras by the PCs. Both productive and nonproductive binding of the chimeras to PC4-PC7 but not to furin contributed to a decreased specificity for inhibiting PC4-PC7 and an increased selectivity for inhibiting furin. Molecular dynamics simulations suggested that nonproductive binding of the chimeras to the PCs was correlated with a greater conformational variability of the catalytic sites of PC4-PC7 relative to that of furin. Our findings suggest a new approach for designing selective inhibitors of PCs using α1PDX as a scaffold, as evidenced by our ability to engineer highly specific and selective inhibitors of furin and PC4-PC7.