Vascular endothelial cells form the inner lining of all blood vessels and play a central role in vessel physiology and disease. Endothelial cells are highly responsive to the mechanical stimulus of fluid shear stress that is exerted by blood flowing over their surface. In this study, the immediate micromechanical response of endothelial cells to physiological shear stress was characterized by tracking of ballistically injected, sub-micron, fluorescent particles. It was found that the mean squared displacement (MSD) of the particles decreases by a factor 1.5 within 10 min after the onset of shear stress. This decrease in particle motion is transient, since the MSD returns to control values within 15–30 min after the onset of shear. The immediate micromechanical stiffening is dependent on activation of the vascular endothelial growth factor receptor (VEGFR)-2, because inhibition of the receptor abrogates the micromechanical response. This work shows that the cytoskeleton is actively involved in the acute, functional response of endothelial cells to shear stress.