TGF--inhibited membrane-associated protein, TIMAP, is expressed at high levels in endothelial cells (EC). It is regarded as a member of the MYPT (myosin phosphatase target subunit) family of protein phosphatase 1 (PP1) regulatory subunits, however, its function in EC is not clear. In our pull-down experiments recombinant TIMAP binds preferentially the isoform of the catalytic subunit of PP1 (PP1c) from pulmonary artery EC. As PP1c, but not PP1c, binds with MYPT1 into functional complex, these results suggested that TIMAP is a novel regulatory subunit of myosin phosphatase in EC. TIMAP depletion by siRNA technique attenuates increases in transendothelial electrical resistance induced by EC barrier-protective agents (sphingosine-1-phosphate, ATP), and enhances the effect of barrier-compromising agents (thrombin, nocodazole) demonstrating a barrier-protective role of TIMAP in EC. Immunofluorescent staining revealed co-localization of TIMAP with membrane/cytoskeletal protein, moesin. Moreover, TIMAP co-immunoprecipitates with moesin suggesting the involvement of TIMAP/moesin interaction in TIMAP-mediated EC barrier enhancement. Activation of cAMP/protein kinase A (PKA) cascade by forskolin, which has a barrier-protective effect against thrombin-induced EC permeability, attenuates thrombin-induced phosphorylation of moesin at the cell periphery of control siRNA-treated EC. On contrary, in TIMAP-depleted EC forskolin failed to affect the level of moesin phosphorylation at the cell edges. These results suggest the involvement of TIMAP in PKA-mediated moesin dephosphorylation and the importance of this dephosphorylation in TIMAP-mediated EC barrier protection.