Human endo-O-sulfatases (Sulf-1 and Sulf-2) are extracellular heparan sulfate proteoglycan (HSPG)-specific 6-O-endosulfatases, which regulate a multitude of cell-signaling events through heparan sulfate (HS)-protein interactions and are associated with the onset of osteoarthritis. These endo-O-sulfatases are transported onto the cell surface to liberate the 6-sulfate groups from the internal D-glucosamine residues in the highly sulfated subdomains of HSPGs. In this study, a variety of HS oligosaccharides with different chain lengths and N-and O-sulfation patterns via chemical synthesis were systematically studied about the substrate specificity of human Sulf-1 employing the fluorogenic substrate 4-methylumbelliferyl sulfate (4-MUS) in a competition assay. The trisaccharide sulfate IdoA2S-GlcNS6S-IdoA2S was found to be the minimal-size substrate for Sulf-1, and substitution of the sulfate group at the 6-O position of the D-glucosamine unit with the sulfonamide motif effectively inhibited the Sulf-1 activity with IC50 = 0.53 mu M, K-i = 0.36 mu M, and K-D = 12 nM.