Abstract: Objective : In the present study, we are attempting to investigate the high fat diet effect on endothelial cell morphology and stiffness. Methods: Rat aorta endothelial cells (RAECs) were obtained from rat thoracic aorta by explants adherent method and identified by factor VIII immunofluorescence staining. Cell morphology was observed under inverted microscopy and quantified by shape parameters including circularity, aspect ratio, perimeter, and area. The mean fluorescent intensity of F-actin was calculated to see the alteration of cell cytoskeleton. Cell stiffness was measured by atomic force microscopy (AFM). Results: The endothelial cells migrated from tissue plant and form confluence after 14days cultivation. The morphology results indicated that cells from high fat diet rats show enhanced perimeter (AS: 145.4±65um vs. CON: 111.9±44um， P＜0.01), aspect ratio (AS: 3.2±1.8 vs. CON: 2.4±1.3，P＜0.01), and area (AS: 554±259um2 vs CON: 542±224um2,P＞0.05), while less circularity (AS: 0.4±0.18 vs. CON: 0.6±0.19，P＜0.01) compared with the control cells. This may be resulted from more F-actin filaments (AS：60±2.9 vs. CON: 50±1.6，P＜0.01) expressed in AS cells. The cell stiffness of the high fat feeding group was significantly higher than that of the normal feeding group (5.2±2.1Kpa VS 4.3±2.2Kpa, p<0.01). Conclusions: High fat diet will change the morphology and stiffness of endothelial cells, which may subsequently affect their normal function, being more vulnerable to atherosclerotic risk factors.