Objectives: Loss of endothelial barrier function leading to oedema formation during reperfusion represents major impediment for the recovery of the organ. The contractility of the endothelial contractile machinery is regulated by the phosphorylation state of the regulatory myosin light chain (MLC), the activity of which is regulated by balanced activities of MLC kinase (MLCK) and MLC phosphatase (MLCP). Reperfusion leads to inhibition of MLCP leading to activation of endothelial contractile machinery and barrier failure. Here, the hypothesis was analyzed that reperfusion leads to activation of endothelial contractile machinery via CPI-17-mediated inhibition of MLCP and inhibition of CPI-17 activates MLCP and barrier stabilisation.Methods: In cultured porcine aortic endothelial cells, the effect of ischemia (45 min, PO2<5 mmHg; pH 6.4) and reperfusion (45 min, PO2=140 mm Hg; pH 7.4) was analyzed on endothelial permeability (albumin flux), contractile activation (MLC phosphorylation), CPI-17 phosphorylation (western blot), MLCP activation (phosphatase assay), actin stress fibre formation and cell-cell adhesions (confocal microscopy).Results: Reperfusion leads to an increase in macromolecule permeability by 150±7 %, MLC phosporylation by 70±7 %, CPI-17 phosphorylation by 210±9 %, and MLCP inactivation by 59±8 % (n 5, P<0.05, for all further parameters). Furthermore, reperfusion lead to an increased actin stress fibre formation and loss of VE-cadherin from cell-cell adhesions. Knockdown of CPI-17 by siRNA resulted an increased basal MLCP activity and significantly abolished reperfusion-induced increase in permeability, MLCP inactivation, MLC phosphorylation, loss of VE-cadherin from cell-cell adhesions and stress fiber formation. Conclusion: Reperfusion activates the endothelial contractile machinery by inhibition of MLCP via activation of CPI-17. Inhibition of CPI-17 presents an important target for reperfusion-induced barrier failure.