The endothelium separates the vascular muscle wall from circulating blood. It is also integrated into complex signaling networks regulating diverse biological processes such as vascular tone, blood flow, barrier as well as fundamental immune responses associated with inflammation and transendothelial migration of circulating leukocytes. The cytoskeletal and intercellular junction proteins of endothelial cells (EC) play an important role in the regulation of barrier integrity, and their physiological functions are mediated by protein kinases and phosphatases. The 20 kDa myosin light chain (MLC20) is phosphorylated on Ser19 and Thr18 sites by myosin light chain kinase during cell contraction and it is dephosphorylated by the myosin phosphatase (PP1M) causing relaxation. One of the potent inhibitor of PP1M function in muscle cells is the smoothelin like protein 1 (SMTNL1). It regulates contraction and relaxation of smooth muscle fibers and plays a central role in vascular and muscle adaptation to exercise and pregnancy by binding to the MYPT regulatory subunit of PP1M. Our goal was to investigate the contribution of SMTNL1 to the endothelial barrier function. We detected the expression of SMTNL1 protein in bovine pulmonary artery endothelial cells (BPAEC) by Western blot analysis. Smtnl1 was silenced by siRNA technique. The phosphorylation level of the inhibitory Thr850 and Thr696 residues of MYPT1 decreased suggesting an increase in the PP1M activity. The phosphorylation of Ser19 residue of MLC20 also decreased. Cell viability of BPEACs reduced by 10-15% and forming of stress fibers was observed by immunofluorescence staining due to SMTNL1 silencing. The adherence ability of siSMTNL1 cells was weaker than that of non-specific siRNA control cells during Electric Cell–substrate Impedance Sensing (ECIS) measurements and the induction of barrier disruption with thrombin and under inflammatory stimulus by TNF resulted in a decrease of the impedance and the resistance of the siSMTNL1 ECs. Based on our results SMTNL1 plays a crucial role in the maintenance of normal barrier function by regulating the actomyosin complex through myosin phosphatase.