Chronic hyperglycemia causes insulin resistance, but the inheritability of glucotoxicity and the underlying mechanisms are unclear. We examined the effect of 3 days of hyperglycemia on glucose disposal, enzyme activities, insulin signaling, and protein O-GlcNAcylation in skeletal muscle of individuals without (FH 2 ) or with (FH + ) family history of type 2 diabetes. Twenty-five subjects with normal glucose tolerance received a [3- 3 H]glucose euglycemic insulin clamp, indirect calorimetry, and vastus-lateralis biopsies before and after 3 days of saline (n = 5) or glucose (n = 10 FH 2 and 10 FH + ) infusion to raise plasma glucose by ∼45 mg/dL. At baseline, FH + had lower insulin-stimulated glucose oxidation and total glucose disposal (TGD) but similar nonoxidative glucose disposal and basal endogenous glucose production (bEGP) compared with FH 2 . After 3 days of glucose infusion, bEGP and glucose oxidation were markedly increased, whereas nonoxidative glucose disposal and TGD were lower versus baseline, with no differences between FH 2 and FH + subjects. Hyperglycemia doubled skeletal muscle glycogen content and impaired activation of glycogen synthase (GS), pyruvate dehydrogenase, and Akt, but protein O-GlcNAcylation was unchanged. Insulin resistance develops to a similar extent in FH 2 and FH + subjects after chronic hyperglycemia, without increased protein O-GlcNAcylation. Decreased nonoxidative glucose disposal due to impaired GS activation appears to be the primary deficit in skeletal muscle glucotoxicity.