Prostaglandin biosynthesis is catalyzed by two spatially and functionally distinct active sites in cyclooxygenase (COX) enzymes. Despite the crucial role of COXs in biology, molecular details regarding the function and regulation of these enzymes are incompletely defined. Reactive nitrogen species, formed during oxidative stress, produce modifications that alter COX functionalities and prostaglandin biosynthesis. We previously established that COX-1 undergoes selective nitration on Tyr385 via a mechanism that requires the presence of bound heme cofactor. As this is a critical residue for COX-1 catalysis, nitration at this site results in enzyme inactivation. We now show that occupancy of the COX-1 active site with substrate protects against Tyr385 nitration and redirects nitration to alternative Tyr residues on COX-1, preserving catalytic activity. This study reveals a novel role for the substrate in protecting COX-1 from inactivation by nitration in pathophysiological settings.