The hexosamine signaling pathway has been shown to serve a nutrient sensing function. We have previously shown that overexpression of the rate limiting enzyme for hexosamine synthesis (glutamine:fructose-6-phosphate amidotransferase, GFA) in adipose tissue of transgenic mice results in skeletal muscle insulin resistance and altered regulation of leptin and adiponectin. In order to dissect the pathways by which the hexosamine pathway affects fuel storage and energy homeostasis, we have examined the characteristics of adipocytes from these animals. After 3 months of age, epididymal fat pads from adult transgenic animals are 42% heavier (p=0.003) and individual adipocytes are 23% larger in diameter (p<0.05) than those from littermate wild type controls. Isolated adipocytes from transgenic mice are insulin resistant, with a 2.5-fold increase in the ED₅₀ for stimulation of 2-deoxy-D-glucose uptake. However, maximal insulin-stimulated glucose uptake is increased in transgenic adipocytes by 39% (p < 0.05). This upregulation of glucose uptake was associated with a 41% increase in the expression of GLUT4 mRNA and a 28% increase in GLUT 4 protein in transgenics compared to controls (p < 0.05). GLUT1 mRNA and protein did not significantly differ between fasted control and transgenics. Total lipid synthesis was also increased in epididymal adipocytes from transgenic animals, by 206% compared to controls (p < 0.05). Fatty acid oxidation was increased 1.6-fold in the transgenic adipocytes (p < 0.05). We conclude that the hexosamine signaling pathway upregulates fat storage in adipocytes in states of carbohydrate excess, in part by increasing GLUT4 and glucose uptake and by augmenting fatty acid synthesis.