Background Posttranslational protein modification with O‐linked N ‐acetylglucosamine (O‐Glc NA c) is linked to high glucose levels in type 2 diabetes mellitus (T2 DM ) and may alter cellular function. We sought to elucidate the involvement of O‐Glc NA c modification in endothelial dysfunction in patients with T2 DM . Methods and Results Freshly isolated endothelial cells obtained by J‐wire biopsy from a forearm vein of patients with T2 DM (n=18) was compared with controls (n=10). Endothelial O‐Glc NA c levels were 1.8‐ford higher in T2 DM patients than in nondiabetic controls ( P =0.003). Higher endothelial O‐Glc NA c levels correlated with serum fasting blood glucose level ( r =0.433, P =0.024) and hemoglobin A _1c ( r =0.418, P =0.042). In endothelial cells from patients with T2 DM , normal glucose conditions (24 hours at 5 mmol/L) lowered O‐Glc NA c levels and restored insulin‐mediated activation of endothelial nitric oxide synthase, whereas high glucose conditions (30 mmol/L) maintained both O‐Glc NA c levels and impaired insulin action. Treatment of endothelial cells with Thiamet G, an O‐Glc NA case inhibitor, increased O‐Glc NA c levels and blunted the improvement of insulin‐mediated endothelial nitric oxide synthase phosphorylation by glucose normalization. Conclusions Taken together, our findings indicate a role for O‐Glc NA c modification in the dynamic, glucose‐induced impairment of endothelial nitric oxide synthase activation in endothelial cells from patients with T2 DM . O‐Glc NA c protein modification may be a treatment target for vascular dysfunction in T2 DM .