Abstract Body: The surge in overweight and obesity rates has triggered a rise in metabolic syndrome cases and related cardiovascular diseases, including vascular stiffness and hypertension. We recently found that consumption of a high-fat/high-fructose Western diet (WD) increases the expression and activation of endothelial cell (EC) mineralocorticoid receptor (ECMR), which is associated with arterial oxidative stress, inflammation, remodeling, and vascular stiffening. However, the precise mechanisms by which enhanced activation of ECMR induces vascular stiffening remain to be elucidated. We hypothesize that miR-99a regulates the activated ECMR-induced upregulation of ECCD36 expression, which promotes vascular endothelial dysfunction and aortic stiffness. First, 16 weeks of WD inhibited miR-99a in the aorta which was prevented by additional spironolactone treatment. With bioinformatics, we predicted that miR-99a would target CD36 expression. To test our hypothesis that miR-99a, regulated by ECMR activation, may also directly affect CD36 mRNA expression in cultured ECs, we used miR-99a mimic or inhibitor to transfect ECs with or without palmitic acid treatment. miR-99a mimic also prevented palmitic acid-induced EC CD36 expression and lipid droplet formation. In vivo gene deletion of ECCD36 blunted WD-induced increases in pulse pressure and reduced vascular activity as well as aortic remodeling. These data indicate that the interaction of endothelial-specific ECMR/miRNA/ECCD36 mediates vascular endothelium dysfunction and prompts arterial stiffness.