There is a current view that myosin light chain kinase (MLCK) plays a critical role in endothelial permeability. To investigate the functions of MLCK in endothelial cells in vivo, we generated a mouse model in which MLCK was selectively deleted by crossing Mylk1 floxed mice with Tie2/cre transgenic mice. Knocking out Mylk1 from endothelial cells had no effect on the global phenotype of the mice, including body weight and blood pressure. Lipopolysaccharide (LPS)‐mediated septic death was also not altered in the knockout (KO) mice. Consistently, LPS‐induced inflammatory injury and the increase in microvascular permeability in the main organs, including the lung and the kidney, was not significantly attenuated in KO mice as compared with wild‐type mice. However, the LPS‐induced microvascular hyperpermeability of the esophagus and the eyeballs was attenuated in KO mice. We also found that the LPS‐mediated increase in the number of caveolae in the endothelial cells of the esophagus was significantly reduced in KO mice. Our results do not support a role for endothelial cell MLCK in the pathogenesis of inflammatory diseases.