Modification of cytosolic, nuclear, and mitochondrial proteins on serine and threonine residues by Nacetylglucosamine (O-GlcNAc) provides a mechanism by which glucose flux rates in the cell can feed back to regulate the function and synthesis of proteins involved in metabolism and growth. The hexosamine biosynthesis pathway (HBP) provides the substrate for this modification, UDP-GlcNAc. Production of UDP-GlcNAc and subsequent protein modifications are generally reflective of glucose flux rates at higher than normal glucose levels, hence the pathway serves a nutrient sensing function. At lower than normal glucose levels, at least some cells upregulate the enzyme responsible for the modification, O-linked GlcNAc transferase (OGT), such that levels of O-GlcNAc on protein also increases at lower than normal glucose concentrations. We summarize herein the evidence that the HBP/O-GlcNAc pathways have an important role in metabolic regulation, both in normal physiology and in pathologic states of high glucose levels. Examples are provided of how the modification alters function of important metabolic enzymes and transcription factors involved in metabolic regulation in muscle, fat, liver, and pancreatic β-cells. The evidence supporting the importance of the pathway is such that any study of metabolic regulation-by mechanisms that are transcriptional, translational, or posttranslational, or through modulation of protein sequestration or degradation-should investigate the possible role of the pathway in that process.