Endothelial cell (EC) survival is critical in the maintenance of endothelial function as well as in the regulation of angiogenesis and vessel integrity since endothelial dysfunction is the initial lesion of atherosclerosis. The goal of this study was to examine the effect of diazoxide, a mitochondrial ATP-sensitive K(+)(mito K(ATP)) channel opener, on aorta ECs apoptosis and its potential mechanism in Otsuka Long-Evans Tokushima Fatty (OLETF) rats at prediabetic stage. Diazoxide (25 mg kg(-1) day(-1)) was administered intraperitoneally from age 8 weeks to age 30 weeks. Thoracic aorta and cultured thoracic aortic ECs were used. The thickening of thoracic aortic wall and apoptosis of ECs were markedly increased in OLETF rats early from the age of 16 weeks, at the impaired glucose tolerance stage, compared with Long-Evans Tokushima Otsuka rats, in conjunction with intimal hyperplasia and perivascular fibrosis. In contrast, diazoxide treatment inhibited these changes. Further study strongly demonstrated that extracellular signal-regulated kinases (ERKs) are key regulatory proteins in protecting ECs from apoptosis. Diazoxide could significantly enhance phosphorylation of ERK via opening mito K(ATP) channels. This role was reversed by both 5-hydroxydecanoate, selectively closing mito K(ATP) channels, and PD-98509, MEK inhibitors. The present studies demonstrate that diazoxide prevents the onset and development of macrovascular disease in OLETF rats by inhibiting apoptosis directly via phosphorylated ERK increase in aorta ECs. Our findings establish the basis for the therapeutic potential of diazoxide in atherosclerotic disease.