Hyperglycemia-induced alterations in mesangial cell function and extracellular matrix protein (ECM) accumulation are seen in diabetic glomerulopathy. The hexosamine biosynthesis pathway (HBP) is implicated in mediating several metabolic effects of high glucose (HG) in cells. This pathway converts fructose-6-phosphate to glucosamine (GlcN)-6-phosphate by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFA). We have previously shown that metabolism of glucose through the HBP regulates the effects of glucose on ECM (fibronectin) synthesis and transcription factor (cyclic adenosine monophosphate-responsive element binding [CREB]) phosphorylation in SV-40-transformed rat kidney mesangial cells. UDP-N-acetyl-GlcN is the end product of the HBP and serves as a precursor for O-linked serine/threonine glycosylation of cytoplasmic and nuclear proteins. Here we show that culturing mesangial cells in HG and GlcN increases the level of O-N-acetylglucosamine in several cytoplasmic and nuclear proteins. Inhibition of O-glycosylation by benzyl-2-acetamido-2-deoxy-α-D-galactopyranoside blocks both HG and GlcN-induced fibronectin synthesis and CREB phosphorylation. To further support the hypothesis that the HBP mediates HG-induced ECM synthesis, a complementary deoxyribonucleic acid (DNA) for human GFA was stably expressed in mesangial cells. Mesangial and GFA-overexpressing cells were cultured in 5 to 25 mM glucose for 48 hours. GFA-overexpressing cells were more sensitive to glucose as they demonstrated increases in fibronectin and CREB phosphorylation at lower glucose concentrations than seen in control cells. In addition, the response to 25 mM glucose for both proteins was increased in GFA when compared with controls. There is no difference in DNA synthesis and cellular adenosine triphosphate levels between the two cell lines. These results suggest that the HBP is a glucose sensor and mediator of the effects of hyperglycemia in the diabetic mesangium.