The cell surface and extracellular matrix polysaccharide, heparan sulfate (HS) conveys chemical information to control or influence crucial biological processes. Attempts to describe its structure-function relationships with HS binding proteins in a classical ‘lock and key’ type manner, however, have been unsuccessful. HS chains are synthesized in a non-template driven process in the ER and Golgi apparatus, involving a large number of enzymes capable of fine-tuning structures. Changes in the localization of HS-modifying enzymes throughout the Golgi, rather than protein expression levels, were found to correlate with changes in the structure of HS. Following brefeldin A treatment, the HS-modifying enzymes localized preferentially in COPII vesicles and at the trans-Golgi. Further, shortly after treatment with heparin, the HS-modifying enzyme moved from cis to trans-Golgi, which coincided with increased HS trisulfated disaccharide content. Finally, it was shown that COPI subunits and Sec24 gene expression changed. Collectively, these findings highlight that the ER-Golgi dynamics of HS-modifying enzymes via vesicular trafficking processes are critical prerequisite for the complete delineation of HS biosynthesis.