Contact between inflammatory cells and endothelial cells (ECs) is a crucial step in vascular inflammation. Recently, we demonstrated that the cell-surface level of endomucin (EMCN), a heavily O-glycosylated single transmembrane sialomucin, interferes with the interactions between inflammatory cells and ECs. We have also shown that, in response to an inflammatory stimulus, EMCN is cleared from the cell surface by an unknown mechanism. In this study, using adenovirus-mediated overexpression of a tagged EMCN in human umbilical vein ECs (HUVECs), we found that treatment with tumor necrosis factor-α (TNF-α) or the strong oxidant pervanadate leads to loss of cell-surface EMCN and increases the levels of the C-terminal fragment of EMCN (EMCN-CTF) 3-4-fold. Furthermore, treatment with the broad-spectrum matrix metalloproteinase (MMP) inhibitor batimastat (BB94) or inhibition of ADAM metallopeptidase domain 10 (ADAM10) and ADAM17 with two small-molecule inhibitors, GW280264X and GI254023X, or with siRNA, significantly reduced basal and TNF-α-induced cell-surface EMCN cleavage. The release of EMCN-CTF by the TNF-α treatment was blocked by chemical inhibition of either ADAM10 alone or in combination with ADAM17. These results indicate that cell-surface EMCN undergoes constitutive cleavage and that TNF-α treatment dramatically increases this cleavage, which is mediated predominantly by ADAM10 and ADAM17. As endothelial cell-surface EMCN attenuates leukocyte-EC interactions during inflammation, we propose that EMCN represents a potential therapeutic target to manage vascular inflammation.,Published under license by The American Society for Biochemistry and Molecular Biology, Inc.