The hexosamine biosynthesis pathway (HBP) is a relatively minor branch of glycolysis. Fructose 6-phosphate is converted to glucosamine 6-phosphate, catalyzed by the first and rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFAT). The major end product is UDP-N-acetylglucosamine (UDP-GlcNAc). Along with other amino sugars generated by HBP, it provides essential building blocks for glycosyl side chains, of proteins and lipids. UDP-GlcNAc regulates flux through HBP by regulating GFAT activity and is the obligatory substrate of O-GlcNAc transferase. The latter is a cytosolic and nuclear enzyme that catalyzes a reversible, posttranslational protein modification, transferring GlcNAc in O-linkage (O-GlcNAc) to specific serine/threonine residues of proteins. The metabolic effects of increased flux through HBP are thought to be mediated by increasing O-GlcNAcylation. Several investigators proposed that HBP functions as a cellular nutrient sensor and plays a role in the development of insulin resistance and the vascular complications of diabetes. Increased flux through HBP is required and sufficient for some of the metabolic effects of sustained, increased glucose flux, which promotes the complications of diabetes, e.g., diminished expression of sarcoplasmic reticulum Ca(2+)-ATPase in cardiomyocytes and induction of TGF-beta and plasminogen activator inhibitor-1 in vascular smooth muscle cells, mesangial cells, and aortic endothelial cells. The mechanism was consistent with enhanced O-GlcNAcylation of certain transcription factors. The role of HBP in the development of insulin resistance has been controversial. There are numerous papers showing a correlation between increased flux through HBP and insulin resistance; however, the causal relationship has not been established. More recent experiments in mice overexpressing GFAT in muscle and adipose tissue or exclusively in fat cells suggest that the latter develop in vivo insulin resistance via cross talk between fat cells and muscle. Although the relationship between HBP and insulin resistance may be quite complex, it clearly deserves further study in concert with its role in the complications of diabetes.