O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. Here we report on regulation of O-GlcNAcylation over a broad range of glucose concentrations. We have discovered a significant induction of O-GlcNAc modification of a limited number of proteins under conditions of glucose deprivation. Beginning 12 h after treatment, glucose-deprived human hepatocellular carcinoma (HepG2) cells demonstrate a 7.8-fold increase in total O-GlcNAc modification compared with cells cultured in normal glucose (5 mm; p = 0.008). Some of the targets of glucose deprivation-induced O-GlcNAcylation are distinct from those modified in response to high glucose (20 mm) or glucosamine (10 mm) treatment, suggesting differential targeting with glucose deprivation and glucose excess. O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation, and this O-GlcNAc increase contributes to a 60% decrease (p = 0.004) in glycogen synthase activity. Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate-limiting substrate for O-GlcNAcylation. Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (OGT) increases 3.4-fold within 6 h of glucose deprivation (p = 0.006). Within 12 h, OGT protein increases 1.7-fold (p = 0.01) compared with normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels (p = 0.03). We conclude that increased O-GlcNAc modification stimulated by glucose deprivation results from increased OGT and decreased O-GlcNAcase levels and that these changes affect cell metabolism, thus inactivating glycogen synthase.