Recently we have shown that cAMP/PKA can protect against thrombin (Thr)-induced endothelial barrier failure by inactivating the contractile machinery via activation of the myosin light chain phosphatase (MLCP). Here the molecular mechanism of MLCP activation by cAMP/PKA and its effect on barrier function were analysed. In HUVEC, activation of adenylyl cyclase by forskolin (FSK) reduced thrombin-induced macromolecule permeability (albumin passage), isometric force (cells cultured on collagen gels), and phosphorylation of myosin light chains. FSK antagonised Thr-induced activation of RhoA and abolished its translocation to membrane. It also induced the assembly and activation of the MLCP complex (recruitment of PP1 catalytic subunit and myosin phosphatase targeting subunit [MYPT1] to myosin; immunoprecipitation and activity assay). Moreover it antagonised Thr-induced phosphorylation of CPI-17, a PP1 inhibitory protein, which led to its detachment from PP1, indicating that the catalytic subunit of the holoenzyme complex is activated. In accordance to this, depletion of CPI-17 by siRNA decreased base line permeability and attenuated Thr-induced hyperpermeability. Conclusion: The data of the present study show that, CPI-17 is involved in modulation of the MLCP holoenzyme activity and is an important target of cAMP/PKAmediated stabilization of endothelial barrier function.