Annexin 1 is an anti-inflammatory protein that plays a key role in innate immunity by modulating the activation of several types of cells including neutrophils. Here we have developed a cleavage assay, using tagged annexin 1, and observed marked activity in the membrane fraction of activated neutrophils. A combination of inhibitors, transfected cells and proteomic analyses, allowed us to identify proteinase 3 as the main enzyme responsible for this cleavage in the N-terminus region of the protein, at least in the context of neutrophil activation. Since annexin 1 is an important endogenous anti-inflammatory mediator, blocking its cleavage by proteinase-3 would augment its homeostatic pro-resolving actions, and could represent an opportunity for innovative anti-inflammatory drug discovery.