Our group and Pierce’s group reported that a brain‐specific β1,6‐N‐acetylglucosaminyltransferase GnT‐IX (GnT‐Vb) has a broad transfer activity toward N‐linked and O‐mannosyl glycans. We showed that its gene expression is regulated by epigenetic histone modifications. We also found that GnT‐IX‐null mice showed enhanced remyelination with impaired astrocyte activation in the cuprizone‐induced demyelination model. Here we show the existence of an endogenous inhibitor for GnT‐IX in Neuro2a (N2a) cell. We purified this inhibitor from N2a cells and found that it is identical to ectonucleotide pyrophosphatase/phosphodiesterase 3 (ENPP3) by mass spectrometry. The underlying mechanism of GnT‐IX inhibition was found to be the ENPP3‐catalyzed hydrolysis of the nucleotide‐sugar donor substrate, UDP‐GlcNAc. Indeed, the ENPP3‐knockdown cells exhibited significantly increased levels of intracellular nucleotide sugars that suggests a biological function of ENPP3 in the regulation of the intracellular nucleotide sugar levels. In addition to chaperones or other known regulators of glycosyltransferases, the ENPP3‐mediated hydrolysis of nucleotide sugars may have significant impacts on other glycosyltransferase activities responsible for altering the total cellular glycosylation profile and modulating cellular functions.