Heparan sulfate (HS) is a complex and diverse polysaccharide abundantly found on the cell surface of cells from several different species. The HS biosynthetic machinery creates very heterogeneous structures that interact with a variety of proteins that result in important biological functions. In addition, HS can be remodeled in the extracellular matrix by a novel class of extracellular sulfatases (Sulfs) which selectively remove 6-O-sulfo groups from glucosamine residues within HS. The activities of Sulfs have been correlated with various biological activities relating to embryonic development and cancer. Therefore, understanding and utilizing Sulf activity can aid in understanding the structure-function relationship of HS as well as aid in developing and tailoring HS based therapies. The goals of this work were to investigate the substrate specificity of human 6-O-endosulfatase isoform 2 (HSulf-2) as well as to utilize HSulf-2 editing activity to tailor anticoagulant HS structures. The use of synthetic polysaccharides along with active HSulf-2 found in the conditioned medium of mammalian cells has allowed for the investigation of the substrate specificity of HSulf-2. Results demonstrated that HSulf-2 is selective toward 6-O-sulfo groups on glucosamine residues that are found in moderately and highly sulfated domains of HS. In addition, 2-O-sulfation of uronic acid and N-sulfation of glucosamine are necessary for serving as a substrate. Results also showed HSulf-2 can tailor HS anticoagulant structures and is capable of maintaining anticoagulant properties while reducing the binding of HS to other proteins. The biochemical characterization of a human heparan sulfate 6-O-endosulfatase as described herein provides a novel method for determining the substrate specificity of HSulf-2. Because Sulf treated HS maintains excellent anticoagulant activity, results of my work open up a new approach to prepare anticoagulant HS with reduced side effects. The future development of this project could elucidate important structure-function relationships as well as become a valuable tool in drug discovery.