The bexosamine biosynthesis pathway (HBP) has been implicated in hyperglycemia-induced insulin resistance. The HBP may also be involved in the development insulin resistance at euglycemia with suppression glucose metabolism (e.g., glycolysis). To evaluate the role of the HBP in fat-induced insulin resistance in vivo, we examined 1) whether fat-induced insulin resistance is additive to that induced by increased HBP flux via glucosamine and, if so, 2) whether such additive effects correlate with muscle HBP product levels. Prolonged (8 h) hyperinsulinemic (-0.6 nM) euglycmic clamps were conducted in conscious overnight fasted rats. After the initial 2.5 h to attain steady-state insulin action, rats received an additional infusion of either saline (n=8). Intralipid (0.9 mL/h; with heparin, 40 U/h; n=9), glucosamine (30 μmol/kg/min; n=8) or Intralipid and glucosamine (n=8) for 5.5 h. At the end of clamps, muscles (soleus, EDL, and tibialis anterior) were taken for the measurement of HBP products. Intralipid and glucosamine infusions decreased insulin action by 38% and 28%, respectively. When the infusions were combined, insulin action decreased 47%, significantly more than with Intralipid or glucosamine infusions alone (P<0.05). Glucosamine-induced insulin resistance was associated with a 4-fold increase in muscle HBP product levels. In contrast, Intralipid-induced insulin resistance was accompanied by absolutely no increase in HBP product levels in all the muscles examined. Also, when infused with glucosamine, Intralipid decreased insulin action below that with glucosamine alone without changing HBP product levels. These data indicate that fat induces insulin resistance independent of the HBP under our experimental conditions.