Hexosamines serve a nutrient-sensing function mediated by enzymatic O-glycosylation of cytosolic and nuclear proteins. We previously characterized transgenic mice with over expression of the rate-limiting enzyme in hexosamine production, GFA, targeted to the beta cell. These animals exhibited hyperinsulinemia that resulted in peripheral insulin resistance. Glucose tolerance deteriorated with age and males developed diabetes. We therefore examined beta cell function by perifusion of isolated islets from these mice. Islets from young (2-month-old) transgenic (Tg) animals had enhanced sensitivity to glucose of insulin secretion. Insulin secretion was maximal at 20 mM glucose and half maximal at 9.9±0.5 mM glucose in Tg islets compared to maximal at 30 mM and half maximal at 13.5±0.7 mM glucose in WT (p<0.005). The 2-month Tg animals secreted more insulin in response to 20 mM glucose (Tg =1254±311, WT=425±231 pg/islet/35 min, p<0.01). Islets from 8-month-old Tg mice became desensitized to glucose, with half-maximal secretion at 16.1±0.8 mM glucose compared to 11.8±0.7 mM in WT (p < 0.05). The 8-month Tg mice secreted less insulin in response to 20 mM glucose (Tg=2256±342, WT=3493±367 pg/islet/35 min, p<0.05). Insulin secretion in response to carbachol was similar in WT and Tg islets at both ages. Glucose oxidation was blunted in the RIP-GFA transgenic islets. At 5 mM glucose, CO₂ production in 8-month islets was comparable between Tg and WT mice. However, islets from C mice increased CO₂ production 2.7±0.4-fold in 20 mM glucose, whereas in Tg islets the increase was only 1.4±0.1-fold (p<0.02). The results demonstrate that hexosamines are involved in nutrient sensing for insulin secretion, and act at least in part by modulating glucose oxidation pathways. Prolonged hexosamine excess flux results in glucose desensitization and mimics glucose toxicity.