Angiopoietin‐like 4 (ANGPTL4) is involved in angiogenesis and lipid metabolism. It is secreted by liver and adipose tissues and cleaved to generate circulating coiled‐coil domain (CCD) and fibrinogen‐like domain (FLD) fragments. The full‐length ANGPTL4 produced by hypoxic endothelial cells interacts with the extracellular matrix (ECM). The ECM‐bound and soluble forms of ANGPTL4 have antiangiogenic properties. We carried out a structure‐function analysis to investigate the regulation of ANGPTL4 bioactivity in endothelial cells. We found that the recombinant CCD binds to the ECM, whereas the FLD is released into the medium. The CCD, like the full‐length ANGPTL4, binds to heparan and dermatan sulfates in surface plasmon resonance assays and inhibits endothelial cell adhesion, motility, and tubule‐like formation. In endothelial cells' ANGPTL4 is processed in the secretion medium after release from the ECM. This processing is altered by the proprotein convertases inhibitor αl‐PDX and abolished by the mutation of the ₁₆₁RRKR₁₆₄ cleavage site without modification of the ECM binding and release. These data suggest that the full‐length form’ which interacts with heparan sulfate proteoglycans via its CCD, is protected from proteolysis by proprotein convertases and constitutes the major active pool of ANGPTL4 in hypoxic endothelial cells.— Chomel, C., Cazes, A., Faye, C., Bignon, M., Gomez, E., Ardidie‐Robouant, C., Barret, A., Ricard‐Blum, S., Muller, L., Germain, S., Monnot, C. Interaction of the coiled‐coil domain with glycosaminoglycans protects angiopoietin‐like 4 from proteolysis and regulates its antiangiogenic activity. FASEB J. 23, 940–949 (2009)