Objective To investigate the high-fat diet effect on morphology and stiffness of endothelial cells. Methods SD rats were randomly divided into high-fat diet group (AS group, n=3) and control group (CON group, n=3). Rat aortic endothelial cells were obtained from rat thoracic aorta by explant method. Cell morphology was observed under inverted microscopy. The mean fluorescent intensity of F-actin in two groups was calculated by immunofluorescence staining. Cell stiffness was measured by atomic force microscopy (AFM). Results The endothelial cells migrated from tissue plant on the 7th day and formed confluence after cultivation for 14 days. Endothelial cells were identified by factor Ⅷ immunofluorescence staining. Cells in AS group showed enhanced perimeter (P＜0.01), aspect ratio (P＜0.01), and area (P＞0.05), while less circularity (P＜0.01) compared with the cells in control group. The mean fluorescence intensity of F-actin in AS group was significantly higher than that in control group (P＜0.01). AFM showed that the cell stiffness of AS group was significantly higher than that of control group (P＜0.01). Conclusions High-fat diet would change the morphology and stiffness of endothelial cells, which might subsequently affect their normal function and become an important incentive to AS.