Heparan sulfate (HS) is a polysaccharide involved in essential physiological functions from regulating cell growth to blood coagulation. HS biosynthesis involves multiple specialized sulfotransferases such as 2-O-sulfotransferase (2OST) that transfers the sulfo group to the 2-OH position of iduronic acid (IdoA) or glucuronic acid (GlcA) within HS. Here, we report the homotrimeric crystal structure of 2OST from chicken, in complex with 3'-phosphoadenosine 5'-phosphate. Structural based mutational analysis has identified amino acid residues that are responsible for substrate specificity. The mutant R189A only transferred sulfates to GlcA moieties within the polysaccharide whereas mutants Y94A and H106A preferentially transferred sulfates to IdoA units. Our results demonstrate the feasibility for manipulating the substrate specificity of 2OST to synthesize HS with unique sulfation patterns. This work will aid the development of an enzymatic approach to synthesize heparin-based therapeutics.