Laskowski inhibitors play vital roles in development, cell differentiation and death by reversibly inhibiting proteolytic enzymes. In nature, these inhibitors masquerade as efficient substrates but they avoid the fate of genuine substrates by way of a unique religation reaction that restores the reactive site bond following cleavage 1,2 . Understanding this mechanism is imperative when adapting these molecules for new protease targets. Here, we use the cyclic peptide, sunflower trypsin inhibitor-1 (SFTI-1)3 , as a model system to explore the molecular basis for Laskowski inhibitor potency and specificity. We demonstrate that pre-organization of the inhibitory loop confers rapid binding association while coordinated inter- and intramolecular interactions promote religation and slow release. By applying these concepts to SFTI-1, we engineered a series of potent inhibitors tailored for selective or broad-range inhibition. Analyzing fold-divergent inhibitor families revealed that these features are generally applicable to Laskowski inhibition, providing new insights on protease inhibitor function and design.