Heparan sulfate (HS) is a linear polysaccharide composed of a glucuronic acid (GlcA)-N-acetyl-glucosamine (GlcNAc) disaccharide repeat motif, polymerized by the EXT1-EXT2 complex. It is extensively modified by a series of Golgi localized enzymes, that generate distinct saccharide sequences involved in the binding and the regulation of numerous protein partners. N-deacetylase/N-sulfotransferase (NDST), of which four isoforms have been identified in mammals, are involved in the first step of this process and catalyze both the N-deacetylation of the GlcNAc residues into GlcNH 2 and its re-N-sulfation into GlcNS residues. Further modifications of the HS chain depend on this first maturation event, NDST action is, therefore, key to HS biosynthesis. However, although the sulfotransferase domain of NDST1 has been characterized at the structural level some 20 years ago, information on the overall structure and activity of the enzyme are still lacking. Here, we report the characterization of the two most expressed NDSTs in humans, NDST1 and NDST2, and a model structure of NDST1 homodimer using cryoelectron microscopy combined with AlphaFold2 modeling. Structure-driven mutagenesis along with two bioassays to follow the protein activities allowed us to characterize the kinetics of the deacetylation and sulfoaddition and to identify the residue H529 as necessary for N-deacetylation. These results shed light on a poorly understood family of enzymes and will help deciphering the molecular basis for HS and heparin maturation.