Remodelling of the extracellular matrix (ECM) plays a crucial role in the development, maintenance and repair of all tissues. Therefore, identifying the regulators of this process is essential for understanding the mechanisms of tissue homeostasis. Among these regulators, A disintegrin and metalloproteinase with a thrombospondin motif 18 (ADAMTS18) has been implicated in fibronectin (FN) matrix regulation. However, how ADAMTS18 influences endothelial specific functions and its role in ECM maturation, particularly in the regulation of FN fibrillogenesis, remains unclear. In this study, using mass spectrometry, we identified two sites in FN that are proteolytically cleaved by ADAMTS18, including a cleavage site in the linker FN-I5-6. Cleavage at this site generated FN molecules lacking the N-terminal FN-I1-5 (29kDa) fragment that is known to be essential for FN fibrillogenesis.