Adducins tightly regulate actin dynamics which is critical for endothelial barrier function. Adducins were reported to regulate epithelial junctional remodeling by controlling the assembly of actin filaments at areas of cell-cell contact. Here, we investigated the role of alpha-adducin for endothelial barrier regulation by using microvascular human dermal and myocardial murine endothelial cells. Parallel transendothelial electrical resistance (TER) measurements and immunofluorescence analysis revealed that siRNA-mediated adducin depletion impaired endothelial barrier formation and led to severe fragmentation of VE-cadherin immunostaining at cell-cell borders. To further test whether the peripheral localization of alpha-adducin is functionally linked with the integrity of endothelial adherens junctions, junctional remodeling was induced by a Ca2+-switch assay. Ca2+-depletion disturbed both linear vascular endothelial (VE)-cadherin and adducin location along cell junctions, whereas their localization was restored following Ca2+-repletion. Similar results were obtained for alpha-adducin phosphorylated at a site typical for PKA (pSer481). To verify that endothelial barrier properties and junction reorganization can be effectively modulated by altering Ca2+-concentration, TER measurements were performed. Thus, Ca2+-depletion drastically reduced TER, whereas Ca2+-repletion led to recovery of endothelial barrier properties resulting in increased TER. Interestingly, the Ca2+-dependent increase in TER was also significantly reduced after efficient alpha-adducin downregulation. Finally, we report that inflammatory mediator-induced endothelial barrier breakdown is associated with loss of alpha-adducin from the cell membrane. Taken together, our results indicate that alpha-adducin is involved in remodeling of endothelial adhesion junctions and thereby contributes to endothelial barrier regulation.