Abnormalities of blood cholesterol concentration are associated with increased risks for vascular disease, especially heart attacks and strokes. As one of the main lipid components of plasma membrane in mammalian cells, cholesterol has a major impact on the mechanical properties of endothelial cells. Although the effect of cholesterol depletion on cell mechanical properties has been studied, no results yet have been reported on quantitative investigation of cholesterol repletion effect. In this study, cholesterol repletion effect on nanomechanical properties of human umbilical vein endothelial cell (EA.hy926) was studied using a control-based atomic force microscope nanomechanical measurement protocol. The viscoelasticity of EA.hy926 cell was measured over a large frequency range (0.1Hz to 100Hz) using both constant-rate excitation force with different loading rates and a broadband excitation force. The experiment results showed that under the cholesterol repletion effect, the Young's modulus and the complex modulus of EA.hy926 cell was increased over 30%, respectively, and moreover, amplitudes of both the elasticity oscillation and the viscosity oscillation at a period around 200 sec were increased over 70%, respectively. Therefore, this work is among the first to investigate the mechanical properties, particularly, the broadband viscoelastic variations of EA.hy926 cells under cholesterol repletion treatment. The results revealed that cholesterol repletion may reinforce the coupling of F-actin to plasma membrane by increasing actin stability, and cholesterol might have modified the submembrane cytoskeletal organization of EA.hy926 cell by causing involvement of the motor protein nonmuscle myosin II.