Fibroblast activation protein-α (FAP) can hydrolyze the post-proline bond. We identified endogenous substrates of FAP in fibroblasts that were previously naive to both FAP and its proteolytic activity. FAP-dependent cleavage sites were identified in many extracellular matrix (ECM) and ECM-associated proteins including collagens and lysyl oxidase-like-1, and CSF-1, CXCL-5 and C1qT6. Quantitative proteomic analysis implicated FAP in ECM-cell interactions, coagulation and metabolism. This study greatly expands the repertoire of FAP substrates and shows that FAP has a role in coagulation in the mouse. Display omitted Highlights •Natural substrates of FAP were identified using degradomic and proteomic techniques and FAP gene knockout mouse derived embryonic fibroblasts stably transduced with enzymatically active or inactive FAP.•Terminal amine isotopic labelling of substrates (TAILS) based degradomics identified cleavage sites in collagens, and many other extracellular matrix (ECM) and associated proteins.•Cleavages of lysyl oxidase-like-1, CXCL-5, CSF-1 and C1qT6 by FAP were confirmed in vitro.•Differential metabolic labelling coupled with quantitative proteomic analysis implicated FAP in regulating proteins that are associated with ECM, ECM-cell interactions, coagulation, metabolism and wound healing. Fibroblast activation protein-alpha (FAP) is a cell-surface transmembrane-anchored dimeric protease. This unique, constitutively active serine protease has both dipeptidyl aminopeptidase and endopeptidase activities and can hydrolyze the post-proline bond. FAP expression is very low in adult organs but is upregulated by activated fibroblasts in sites of tissue remodeling, including fibrosis, atherosclerosis, arthritis and tumors. To identify the endogenous substrates of FAP, we immortalized primary mouse embryonic fibroblasts (MEFs) from FAP gene knockout embryos and then stably transduced them to express either enzymatically active or inactive FAP. The MEF secretomes were then analyzed using degradomic and proteomic techniques. Terminal amine isotopic labeling of substrates (TAILS)-based degradomics identified cleavage sites in collagens, many other extracellular matrix (ECM) and associated proteins, and lysyl oxidase-like-1, CXCL-5, CSF-1, and C1qT6, that were confirmed in vitro. In addition, differential metabolic labeling coupled with quantitative proteomic analysis also implicated FAP in ECM-cell interactions, as well as with coagulation, metabolism and wound healing associated proteins. Plasma from FAP-deficient mice exhibited slower than wild-type clotting times. This study provides a significant expansion of the substrate repertoire of FAP and provides insight into the physiological and potential pathological roles of this enigmatic protease.