p53 is a guardian of the genome that protects against carcinogenesis. There is accumulating evidence that p53 is activated with aging. Such activation has been reported to contribute to various age-associated pathologies, but its role in vascular dysfunction is largely unknown. The aim of this study was to investigate whether activation of endothelial p53 has a pathological effect in relation to endothelial function. We established endothelial p53 loss-of-function and gain-of-function models by breeding endothelial-cell specific Cre mice with floxed Trp53 or floxed Mdm2/Mdm4 mice, respectively. Then we induced diabetes by injection of streptozotocin. In the diabetic state, endothelial p53 expression was markedly up-regulated and endothelium-dependent vasodilatation was significantly impaired. Impairment of vasodilatation was significantly ameliorated in endothelial p53 knockout (EC-p53 KO) mice, and deletion of endothelial p53 also significantly enhanced the induction of angiogenesis by ischemia. Conversely, activation of endothelial p53 by deleting Mdm2/Mdm4 reduced both endothelium-dependent vasodilatation and ischemia-induced angiogenesis. Introduction of p53 into human endothelial cells up-regulated the expression of phosphatase and tensin homolog (PTEN), thereby reducing phospho-eNOS levels. Consistent with these results, the beneficial impact of endothelial p53 deletion on endothelial function was attenuated in EC-p53 KO mice with an eNOS-deficient background. These results show that endothelial p53 negatively regulates endothelium-dependent vasodilatation and ischemia-induced angiogenesis, suggesting that inhibition of endothelial p53 could be a novel therapeutic target in patients with metabolic disorders.