In blood vessels, nitric oxide (NO) is the main vasodilator. NO production critically depends on the plasticity of the endothelial cell cortex (eCC), the ~100 nm thick layer beneath the plasma membrane which changes from soft to stiff if necessary. Nevertheless, it is still unknown what exactly determines the elasticity of this functional layer. One hypothesis is that a decrease of eCC stiffness involves the dynamics of the submembranous F-actin meshwork. However, a direct proof is missing whether mechanics of the eCC are directly related to the function of the submembrane actin web. To visualize cortical F-actin dynamics in living endothelial cells we transfected cells with Lifeact, a small fluorescent protein that associates with F-actin without functional disturbances. Cortical plasticity was measured by nanoindentation using atomic force microscopy. To analyze simultaneously the relationship of F-actin organization and cortical plasticity, we applied low doses (50 nM) of Cytochalasin D (CytD). This treatment ensures that only the F-actin within the first 100 nm below the cell membrane is destabilized. CytD induced a decrease of cortical stiffness by ~40% whereas the stiffness of the cell centre remained unchanged. Simultaneously, Lifeact fluorescence intensity decreased by ~20%. Analyses indicate a negative correlation of eCC plasticity and Lifeact fluorescence (R² = 0.85). Additionally, simultaneous NO and Lifeact measurements reveal that cortical actin depolymerization induces endothelial NO release. We conclude that changes of eCC plasticity directly depend on cortical actin organization, which is a major determinant of NO release and thus blood vessel tone.