Endothelial barrier dysfunction is a key event in the pathogenesis of atherosclerosis. The objective of this study was to explore the role and mechanisms of MLCK210 (non‐muscle myosin light‐chain kinase) in the development of atherosclerosis in mice with an apoE knockout background. Histopathological examination demonstrated that MLCK210 deficiency reduced atherosclerotic lesion and macrophage deposit. Western blot and RT‐PCR analyses revealed MLCK210 expression in the aortic endothelium and peripheral monocytes. By measuring transendothelial electric resistance, we found that MLCK210 deficiency attenuated aorta endothelial hyperpermeability to pro‐inflammatory stimuli, including thrombin, ox‐LDL and TNF‐alpha. In monocytes, MLCK210 deficiency reduced their transendothelial migration in response to murine MCP‐1. Further mechanistic studies showed that these inflammatory mediators induced Src phosphorylation in endothelial cells and monocytes. The phosphorylation response was attenuated in MLCK210 deficient cells, corresponding to reduced endothelial permeability and monocytes transmigration. Taken together, the data suggest that MLCK210 contributes to the development of atherosclerosis by regulating endothelial barrier function and monocyte migration via a mechanism involving Src signaling. Supported by NIH HL61507, HL84542, HL70752, and HL73324.