Endothelial dysfunction (ED) and hyperpermeability are considered as the initiating steps in early atherosclerosis. Phosphorylation of myosin light chain (MLC) is key to cause vascular hyperpermeability via endothelial cell contraction. However, it is unclear whether MLC phosphorylation can also regulate the balance between contraction and relaxation of endothelial cells, thereby affecting endothelium-dependent diastolic function and leading to ED. The present study investigated relationships between ED and MLC phosphorylation and underlying mechanisms. Twenty-four male New Zealand white rabbits were randomly divided into three groups: control, AS, and ML7 (MLCK inhibitor) groups, and fed with normal diet, high-fat diet (HFD), and HFD plus oral ML7 (1 mg/kg daily) respectively. HFD-fed rabbits showed typical atheromatous lesions and endothelial hyperpermeability, and these lesions could be partly reversed following ML7 therapy. Western blotting revealed significant increased expression of myosin light chain kinase (MLCK) and phosphorylation of MLC, JNK, and ERK in the arterial wall of rabbits in the AS group compared with those of the control group (p < 0.05), whereas the ML7 group showed markedly decreased levels of these proteins compared with the AS group (p <  0.05). The endothelium-dependent relaxation rate was significantly reduced both in vitro and in vivo in AS group, and was improved using ML7 therapy. Taken together, these results indicate that MLCK expression and subsequent MLC phosphorylation increase vascular endothelial permeability and endothelium-dependent diastolic dysfunction by promoting endothelial cell contraction, which may be initiated by the activation of the MAP/ERK (MEK) and MAP/JNK (MEK) pathways.