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Heparan sulfate 3- O -sulfotransferase (3-OST) transfers a sulfo group to the 3-OH position of a glucosamine saccharide unit to form 3- O -sulfated heparan sulfate. 3- O -sulfation is known to be critically important for bestowing anticoagulant activity and other biological functions of heparan sulfate. Here, we report two ternary crystal structures of 3-OST-5 with PAP (3'-phosphoadenosine 5'-phosphate) and two octasaccharide substrates. We also used 3-OST-5 to synthesize six 3- O -sulfated 8-mers. Results from the structural analysis of the six 3- O -sulfated 8-mers revealed the substrate specificity of 3-OST-5. The enzyme prefers to sulfate a 6- O -sulfo glucosamine saccharide that is surrounded by glucuronic acid over a 6- O -sulfo glucosamine saccharide that is surrounded by 2- O -sulfated iduronic acid. 3-OST-5 modified 8-mers display a broad range of anti-factor Xa activity, depending on the structure of the 8-mer. We also discovered that the substrate specificity of 3-OST-5 is not governed solely by the side chains from amino acid residues in the active site. The conformational flexibility of the 2- O -sulfated iduronic acid in the saccharide substrates also contributes to the substrate specificity. These findings advance our understanding for how to control the biosynthesis of 3- O -sulfated heparan sulfate with desired biological activities., Competing Interests: Competing interest RW, AMK, ZW, ES, Jine Li, JMK and LCP declare no competing interest.