Impaired insulin receptor (IR) activity has been found in various models of insulin resistance, including models of injury or critical illness, and Type 2 diabetes. However, mechanisms that modulate IR function remain unclear. With an animal model of critical illness diabetes, we found insulin-induced IR tyrosine phosphorylation in the liver was impaired as early as 15 minutes following trauma and hemorrhage. Possible mechanisms for this defect were examined, including IR protein levels and IR post-translational modifications. The total amounts of hepatic IR α and β subunits and the membrane localization of the IR were not altered by trauma and hemorrhage, and, likewise, no change in IR tyrosine nitration was found in the liver. However, there was a decrease in the level of protein O-linked beta-N-acetlyglucosamine (O-GlcNac) modification on Ser/Thr in the liver following trauma and hemorrhage. Inhibition of c-jun N-terminal kinase (JNK) increased IR O-GlcNac modification implicating an involvement of JNK. These findings suggest that a balance between O-GlcNac modification and JNK-induced phosphorylation may exist, with decreased Ser/Thr O-GlcNac modification following trauma and hemorrhage, allowing JNK to phosphorylate the IR on neighboring Ser/Thr residues, which subsequently inhibits IR activity. The present studies suggest potential mechanisms of hemorrhage-induced defects in IR activity, and a potential role for acutely decreased O-GlcNac and increased serine phosphorylation of the IR.