Ischemic heart disease is the leading cause of death in diabetic patients and coronary endothelial dysfunction plays a crucial role in the development and progression of ischemic heart disease. Excessive protein O-GlcNAcylation is observed in diabetic patients and it contributes to the functional changes in many tissues. To define the role of protein O-GlcNAcylation in coronary endothelial functions in diabetes, we used our unique transgenic mouse, endothelial cell (EC)-specific tet-inducible O-GlcNAcase (OGA, an enzyme which decreases protein O-GlcNAcylation) mouse and crossed it with TALLYHO mouse (TH mouse, a polygenic model for type 2 diabetes). TH mice exhibited increased apoptotic ECs and reduced capillary density in the left ventricular (LV) compared to the wild-type (WT) mice. Myocardial infarct size after ischemia/reperfusion was increased and cardiac functions were attenuated in TH mice compared to WT mice. Mouse coronary ECs (MCECs) isolated from TH mice showed higher protein O-GlcNAcylation than control MCECs. Overexpression of OGA in TH mice not only increased capillary density in the LV, but also restored cardiac contractility toward the level in WT mice. High-glucose (HG) treatment in human coronary ECs (HCECs) led to cell apoptosis and attenuated tube formation. OGA overexpression in HG-treated HCECs decreased protein O-GlcNAcylation and increased tube formation. It has been reported that O-GlcNAcylation of p53 increases p53 stability and its activity. p53 protein expression was significantly increased by HG treatment and decreased by OGA overexpression. p53 is a transcription factor and its activation induces cell apoptosis. MCECs isolated from TH mice showed higher p53 protein expression than MCECs in WT mice. Chronic treatment of Pifithrin-α (PFT, an inhibitor of p53) in TH mice significantly increased capillary density in the LV, and restored cardiac contractility toward to the level in WT mice. We examined downstream targets of p53 using a PCR array and found that Bax, PUMA and TNF are the potential targets regulated by p53 in TH mice.These data suggest that O-GlcNAcylation of p53 leads to coronary endothelial dysfunction and results in attenuated cardiac contractility in diabetes.