Heparan sulfate (HS), a universally expressed and abundant macromolecule found at the cell surface and in the extracellular matrix, plays fundamental roles in a wide variety of physiological and pathophysiological processes, and it represents a promising therapeutic agent for current and future pharmaceutical research. The diverse functions of HS rely on its diverse structures, resulting from modifications by many HS biosynthetic enzymes. Understanding HS fine structure regulation by these enzymes is fundamental to therapeutic intervention targeting numerous HS involved diseases. Towards this end, characterization of all HS modification enzymes and their structure-function relationships is essential. In this research, the substrate specificity of 3OST6, a novel HS 3-O-sulfotransferase, was characterized using a CHO cell line stably expressing 3OST6. The HS structure generated by 3OST6 was also determined to be capable of serving as an HSV-1 entry receptor. Next, facilitated by a novel recombinant protein expression system developed in the lab, HS 3OST5 was purified and its crystal structure was solved in complex with 3′- phosphoadenosine 5′-phosphate; while HS 2OST was purified and is currently under crystallization trials. Moreover, a systematic mutational study of HS 2OST and chondroitin sulfate (CS) 2OST, an enzyme homologous to HS 2OST modifying CS chain, was conducted based on homology models derived from structure-solved sulfotransferases. From this study, a co-factor binding site and a potential substrate binding site were identified. More importantly, a unique catalytic site of HS 2OST was proposed, which is not present in CS 2OST even though these two enzymes catalyze the same chemical reaction. This study provided the first structure-function relationship data for these two enzymes. Furthermore, it prompted us to identify the authentic enzyme possessing the function of HS 2OST in Drosophila, which was mistakenly assigned to another homologous protein.