MafA is a basic leucine zipper transcription factor that regulates gene expression in both the neuroretina and pancreas. Within the pancreas, MafA is exclusively expressed in the beta cells and is involved in insulin gene transcription, insulin secretion, and beta cell survival. The expression of the mafA gene within beta cells is known to increase in response to high glucose levels by an unknown mechanism. In this study, we demonstrate that pyruvate, which is produced by glycolysis from glucose, is not sufficient to induce mafA gene expression compared with high glucose. This suggests that the signal for MafA induction is independent of ATP levels and that a metabolic event occurring upstream of pyruvate production leads to the induction of MafA. Furthermore, insulin secretion mediated by high glucose is not important for MafA expression. However, the addition of glucosamine to beta cell lines stimulates MafA expression in the absence of high glucose, and inhibition of the hexosamine biosynthetic pathway in the presence of high glucose abolishes MafA induction. Moreover, we demonstrate that the expression of UDP-N-acetylglucosaminyl transferase, the enzyme mediating O-linked glycosylation of cytosolic and nuclear proteins, is essential for glucose-dependent MafA expression. Consistent with this observation, inhibition of N-acetylglucosaminidase, the enzyme involved in the removal of the O-GlcNAc modification from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate stimulates MafA expression under low glucose conditions. The presented data suggest that MafA expression mediated by high glucose requires flux through the hexosamine biosynthetic pathway and the O-linked glycosylation of an unknown protein(s) by UDP-N-acetylglucosaminyl transferase.