Type 2 diabetes mellitus (T2DM) afflicts ~25 million people in the United States and hyperglycemia is a major causative factor contributing to vascular dysfunction among this population. This study evaluated the hypothesis that hyperglycemia-induced changes in endothelial glycoproteome leads to alterations of important homeostatic signaling pathways that result in vascular dysfunction. Utilizing targeted glycoprotein enrichment coupled with tandem mass spectrometry for protein identification and quantification, the cell surface glycoproteins were assessed from rat microvascular endothelial cells (RMVECs) cultured in ‘normal’ glucose (5 mM) or ‘high’ glucose (25 mM). Altogether, 273 N-glycosylation modified proteins were identified; 65 proteins uniquely N-glycosylated and 22 proteins significantly increased in N-glycosylation (p<0.05) from ‘high’ glucose RMVECs. Additionally, 499 O-glycoproteins were identified; 78 proteins uniquely O-glycosylated and 65 proteins significantly increased in O-glycoslyation (p<0.05) from ‘high’ glucose RMVECs. The type-1A angiotensin II receptor (AT1; p=0.029), along with numerous cell adhesion molecules, had increased N-glycosylation in ‘high’ glucose RMVECs. Numerous ion channels important for vascular function and the 5-HT2A serotonin receptor (p=5.3e-28), recently shown to contribute to vascular dysfunction in T2DM, had elevated O-glycosylation in ‘high’ glucose RMVECs. In order to evaluate these targets, functional assays were developed to measure endothelial and vascular function. Immobilization assays in a parallel plate flow chamber (PPFC) indicated that ‘high’ glucose significantly increased RMVEC adhesion to gelatin substrate under laminar flow (p<0.05). In addition, incubation of RMVECs under ‘high’ glucose conditions increased the adherence of healthy platelets to RMVECs, suggesting the importance of adhesion protein glycosylation in endothelial dysfunction. Finally, studies in human subcutaneous arterioles demonstrated that acute exposure to ‘high’ glucose impaired acetylcholine-induced vasodilation. Utilizing these assays, differentially hyperglycemia-regulated targets will be functionally evaluated for potential therapeutic intervention.