Molecular rotors are fluorescent probes that change quantum yield with the viscosity of their environment. When integrated into the cell membrane, they can be used to probe viscosity changes of the membrane. Fluid shear stress is hypothesized to increase membrane fluidity in the membrane of endothelial cells, a change that leads to the activation of heterotrimetric G proteins, thus activating a signal transduction cascade. This hypothesis was examined using a molecular rotor, 9-dicyanovinyl-julolidine (DCVJ) as membrane probe. The principal response, a decease of fluorescence intensity caused by increased membrane fluidity, was obtained by adding a fluidity-increasing agent to the cells. In a parallel-plate flow chamber, a confluent layer of DCVJ-labeled human umbilical cord venous endothelial cells were exposed to different levels of fluid shear stress. With increased shear, a reduced fluorescence intensity was observed, indicating an increase of membrane fluidity. Step changes of fluid shear stress caused an approximately linear drop of fluorescence within 5 seconds, showing fast and almost full recovery after shear stopped. A linear relationship between shear stress and membrane fluidity changes was also observed. This study not only shows the suitability of the molecular rotor DCVJ as a membrane fluidity probe, but also provides evidence for the direct link between fluid shear stress and membrane fluidity, and suggests that the membrane is the primary flow mechanosensor of the cell.