Decoy receptor 3 (DcR3), a soluble receptor for Fas ligand, LIGHT (homologous to lymphotoxins shows inducible expression and competes with HSV glycoprotein D for herpes virus entry mediator, a receptor expressed by T lymphocytes), and TNF-like molecule 1A, is highly expressed in cancer cells and in tissues affected by autoimmune disease. DcR3.Fc has been shown to stimulate cell adhesion and to modulate cell activation and differentiation by triggering multiple signaling cascades that are independent of its three known ligands. In this study we found that DcR3.Fc-induced cell adhesion was inhibited by heparin and heparan sulfate, and that DcR3.Fc was unable to bind Chinese hamster ovary K1 mutants defective in glycosaminoglycan (GAG) synthesis. Furthermore, the negatively charged, sulfated GAGs of cell surface proteoglycans, but not their core proteins, were identified as the binding sites for DcR3.Fc. A potential GAG-binding site was found in the C-terminal region of DcR3, and the mutation of three basic residues, i.e., K256, R258, and R259, to alanines abolished its ability to trigger cell adhesion. Moreover, a fusion protein comprising the GAG-binding region of DcR3 with an Fc fragment (DcR3_HBD.Fc) has the same effect as DcR3.Fc in activating protein kinase C and inducing cell adhesion. Compared with wild-type THP-1 cells, cell adhesion induced by DcR3.Fc was significantly reduced in both CD44v3 and syndecan-2 knockdown THP-1 cells. Therefore, we propose a model in which DcR3.Fc may bind to and cross-link proteoglycans to induce monocyte adhesion.