Context:Because current tests available for the diagnosis of diabetes have shortcomings, a novel screening method for the earlier and more efficient detection of type 2 diabetes would be a significant clinical advance.Objective:The hexosamine biosynthetic pathway usually acts as a fuel sensor, and its activation leads to O-linked β-N-acetylglucosamine (O-GlcNAc) modification of target proteins (O-GlcNAcylation) in a glucose-responsive manner. O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are responsible for O-GlcNAc addition and removal, respectively. Because higher hexosamine biosynthetic pathway flux is linked to insulin resistance/type 2 diabetes, we hypothesized that increased O-GlcNAcylation of leukocyte proteins can detect the onset of pre- and overt diabetes.Design, Setting and Patients:Seventy-four participants from Bellville and Stellenbosch (Western Cape, South Africa) were recruited and classified as normal, prediabetic, and diabetic individuals (American Diabetes Association criteria).Main Outcome Measures:Leukocytes isolated from study subjects were evaluated for O-GlcNAc, OGA, and O-GlcNAc transferase expression by flow cytometry and immunofluorescence microscopy.Results:Flow cytometric analysis of leukocyte subtypes revealed increased O-GlcNAcylation in granulocytes vs. lymphocytes (P < 0.001). Diabetic individuals displayed higher leukocyte O-GlcNAcylation (P < 0.01), whereas granulocyte analysis showed an increase for prediabetic subjects (P < 0.01). However, OGA expression increased in leukocytes of diabetic subjects and is likely an adaptation to attenuate higher O-GlcNAcylation observed (P < 0.001).Conclusions:Together our data demonstrate that leukocyte (particularly granulocyte) O-GlcNAcylation could help detect pre- and overt diabetes and offer clinical value as unique markers for the earlier and more efficient detection of type 2 diabetes.