Heparan sulfate (HS) is an abundantpolysaccharide in the animal kingdom withessential physiological functions.HS is composed of sulfated saccharides that arebiosynthesized through a complex pathwayinvolving multiple enzymes.In vivoregulation of this process remains unclear.HS 2-Osulfotransferase (2OST) is a key enzyme in thispathway.Here,we report the crystal structure of the ternary complex of 2OST,3′-phosphoadenosine 5′-phosphate,and a heptasaccharide substrate.Utilizing site-directed mutagenesis and specificoligosaccharide substrate sequences,we probedthe molecular basis of specificity and 2OSTsposition in the ordered HS biosynthesis pathway.These studies revealed that Arg80,Lys350,and Arg190 of 2OST interact with the N-sulfo groups near the modification site,consistent with the dependence of 2OST on N-sulfation.In contrast,6-O-sulfo groups on HS are likely excluded by steric and electrostaticrepulsion within the active site supporting the hypothesis that 2-O-sulfation occurs prior to 6-O-sulfation.Our results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.