Introduction: Western diet (WD) associates with elevations in blood lipids. Endothelial cells (EC) uptake lipids through scavenger receptors such as CD36. In our studies we demonstrated that exposure to lipids induces a stiffening response in ECs, with in vivo studies showing that this stiffening is abrogated in mice with a global CD36 knockout. Hypothesis: WD associates with aortic barrier disruption. in vitro studies have demonstrated that EC stiffening correlates with barrier disruption. It is our hypothesis that EC stiffening mediates WD-induced aortic barrier disruption in an endothelial CD36-dependent manner. Methods: To generate a model of endothelial-specific CD36 deficiency, we crossed tamoxifen-inducible Cre mice with an EC-promoter with CD36 fl/fl mice. Cre only mice are employed as controls. At the end of the study window, aortas are excised for assessment of EC stiffening via atomic force microscopy. Separate cohorts are assessed for barrier disruption via quantification of Evans blue dye (EBD) leakage or VE-cadherin junctional thickness. Inhibition of CD36 in vitro was accomplished with SSO. For cell experiments we employed long-chain fatty acids (LCFAs), lipid ligands for CD36. Results: WD induced significant stiffening of the aortic endothelium [p<0.05]. This phenomenon was only observed in male (n=5-6) and not female (n=3-10) mice. EC stiffening associated with aortic barrier disruption as indicated by increased EBD permeation (n=3-8) and increased VE-cadherin thickness (n=3-4) [p<0.05]. Use of an endothelial-specific CD36 deficiency significantly abrogated stiffening and barrier disruption [p<0.05]. Controls demonstrated no effects by tamoxifen (n=2-3). Exposure to 5 μM of palmitic and stearic acids (saturated LCFAs) results in stiffening while exposure to oleic acid (unsaturated LCFA) does not (n=5-12) [p<0.05]. Palmitic acid-induced EC stiffening was shown to be dependent on CD36 (n=5-6) and activation of RhoA protein kinase (n=3-8) [p<0.05]. Conclusions: Our results demonstrate that endothelial CD36 is required for WD-induced EC stiffening and subsequent disruption of the aortic barrier. Furthermore, our results suggest that CD36 mediates EC stiffening through its role as scavenger receptor to saturated LCFAs.