The human form of UDP-xylose synthase (hUXS1A) is studied with respect to its substrate and co-enzyme binding in binary and ternary complexes using saturation transfer difference (STD) NMR and in situ NMR. Obtained binding pattern results are correlated to the recently solved crystal structure of hUXS1A and docking studies of UDP-GlcUA, providing a better understanding of substrate specificity of this enzyme and may give useful information in mutant designing. In unproductive binary complexes UDP-saccharide aglycone moieties show strong STD effects with the protein. In contrast, pyranoside rings (Glc, GlcUA, Gal) indicate less interaction with the hUXS1A active site, which enables the required ring distortion of the pyranoside ring in UDP-GlcUA. In productive ternary complexes UDP-GlcUA possesses reasonable binding, while produced UDP-Xyl shows smaller STD responses and does not efficiently compete with the substrate for binding at the active site. STD NMR derived binding studies of NAD+ demonstrate tight interaction between co-factor and hUXS1A. Higher magnetization of NAD+ in the presence of enzymatic product is observed and suggests increased contact with groups on the protein. Furthermore, binding studies of substrate analogues having the same stereochemistry as the investigated UDP-saccharides and a small aglycone residue indicate a different mode of action, not guided by the anchor groups.