Several endothelial functions are affected by fluid shear stress, but the mechanisms underlying the adaptation of paracellular barrier function are unknown. Recently we described that unidirectional shear stress induced a rac1-dependent transient increase in endothelial barrier function in the first minutes followed by an adaptation in the next hours. This adaptation is associated with an elongation of the cells, which in turn increases the length of the cell borders and decreases the paracellular barrier function moderately. Immunofluorescence and Western Blot analysis showed intact cell-cell contacts, actin-recruitment to the junctions and enhanced expression of VE-cadherin and catenins indicating counter regulatory mechanisms. Inhibition of rac1 blocked cell elongation, actin recruitment and the increased expression of junctional proteins, resulting in a breakdown of endothelial barrier function under shear stress. We conclude that rac1 plays a critical role during adaptation of endothelial barrier function via regulation of protein expression, junctional reorganisation and morphological alterations.