Abstract <p>Actin is one of the most abundant proteins, it presents in a living cell in different isoforms depending on the cell type. β-actin (β-Cya) and γ-actin (γ-Cya) are cytoplasmic actin isoforms in non-muscle mammalian cells and their spatial arrangement provides their divers roles in cellular functions. In this work, we used 3D-SIM-microscopy to investigate for the first time β/γ-Cya colocalization in endothelial cells (EC), essential for EC barrier function implementation. We demonstrated β/γ-Cya partial colocalization in certain regions of EC cytoplasm, varies widely between different parts of the marginal regions and near the cell nucleus. In the basal cytoplasm, β-Cya predominates, while the ratio of isoforms evens out as it moves to the apical cytoplasm. Thus, colocalization analysis suggests that β- and γ-Cya are segregated in the endotheliocyte cytoplasm. This segregation is greatly enhanced during EC activation in experiments, modeling endothelial barrier dysfunction in vitro, demonstrating different roles of β/γ-actins in the EC functional activity.</p>