O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. We have previously shown a significant induction of O-GlcNAc modification under conditions of glucose deprivation. Increased O-GlcNAc modification was mediated by increased mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (ncOGT). We have investigated the mechanism mediating ncOGT induction with glucose deprivation. The signal does not appear to be general energy depletion because no differences in AMP-dependent kinase protein levels or phosphorylation were observed between glucose-deprived and normal glucose-treated cells. However, treatment of glucose-deprived cells with a small dose (1 mm) of glucosamine blocked the induction of ncOGT mRNA and subsequent increase in O-GlcNAc protein modification, suggesting that decreased hexosamine flux is the signal for ncOGT up-regulation. Consistent with this, treatment of glucose-deprived cells with an inhibitor of O-GlcNAcase (O-(2-acetamido-2-deoxy-D-glucopyranosylidene) amino N-phenyl carbamat) completely prevented the subsequent up-regulation of ncOGT. Glucosamine treatment also resulted in a 40% rescue of the down-regulation of glycogen synthase activity normally seen after glucose deprivation. We conclude that deglycosylation of proteins within the first few hours of glucose deprivation promotes ncOGT induction. These findings suggest a novel negative feedback regulatory loop for OGT and O-GlcNAc regulation.