The endothelial monolayer is located on the inner surface of blood vessels and provides an important barrier function controlling the transport of metabolites and nutrients through the vessel wall, both toward the circulating blood and in the direction of the underlying tissues. This function is provided by cytoskeletal structures that generate contractile and tensile forces, existing in equilibrium in the intact endothelium. In the case of cytoskeleton rearrangements, there are changes in the shape of cells and the formation of intercellular spaces, which lead to endothelial dysfunction. Deep understanding of endothelial barrier function maintaining is a crucial problem because this phenomenon is common for a number of pathological states and diseases (inflammation, asthma, sepsis, acute lung injury, diabetes, etc.) and can lead to severe organ dysfunction, as well as a complication upon the treatment by a number of anticancer pharmacological drugs. Microtubules and the actin cytoskeleton function in cooperation in normal endothelium and under conditions of the barrier loss. In this review, we describe the application of modern fluorescence methods for investigation and analysis of the individual characteristics of cytoskeletal elements whose reorganization affects endothelial permeability, to emphasize the role of microtubules/microfilament crosstalk in EC barrier regulation.