Alignment of vascular endothelial cells in the direction of the flow is considered to be a key factor in maintaining endothelial integrity in active hemodynamic environment. Our recent studies have shown that exposure to oxLDL, one of the major pro-atherogenic lipoproteins, significantly increases the stiffness of human aortic endothelial cells (HAECs) suggesting that oxLDL may have a significant impact on the sensitivity of endothelial cells to mechanical stimuli. In this study, we show that oxLDL strongly enhances the ability of endothelial cells to realign in the direction of the flow and facilitates the formation of F-actin stress fibers both under static and under flow conditions. The impact of oxLDL on the flow-induced realignment is observed on both whole cell and single fibers levels. We also show that similarly to the effect of oxLDL on endothelial stiffness, its impact on flow-induced realignment can be simulated by MCD-induced cholesterol depletion supporting the hypothesis that oxLDL acts as cholesterol acceptor rather than cholesterol donor for endothelial cells. Finally, we propose that oxLDL/cholesterol depletion-induced sensitization of endothelial cells to flow may be a result of an increase in cellular stiffness and a respective increase in membrane-cytoskeleton tension.