Vascular endothelial dysfunction (VED) is an important factor in the initiation and development of atherosclerosis (AS). Previous studies have demonstrated that endothelial permeability is increased in diet‑induced AS. However, the precise underlying mechanisms remain poorly understood. The present study aimed to analyze whether the myosin light chain kinase (MLCK) inhibitor ML7 is able to improve VED and AS by regulating the expression of the tight junction (TJ) proteins zona occludens (ZO)‑1 and occludin via mechanisms involving MLCK and MLC phosphorylation in high‑fat diet‑fed rabbits. New Zealand white rabbits were randomly divided into three groups: Control group, AS group and ML7 group. The rabbits were fed a standard diet (control group), a high‑fat diet (AS group) or a high‑fat diet supplemented with 1 mg/kg/day ML7 (ML7 group). After 12 weeks, endothelium‑dependent relaxation and endothelium‑independent relaxation were measured using high-frequency ultrasound. Administration of a high‑fat diet significantly increased the levels of serum lipids and inflammatory markers in the rabbits in the AS group, as compared with those in the rabbits in the control group. Furthermore, a high‑fat diet contributed to the formation of a typical atherosclerotic plaque, as well as an increase in endothelial permeability and VED. These symptoms of AS were significantly improved following treatment with ML7, as demonstrated in the ML7 group. Hematoxylin & eosin and immunohistochemical staining indicated that ML7 was able to decrease the expression of MLCK and MLC phosphorylation in the arterial wall of rabbits fed a high‑fat diet. A similar change was observed for the TJ proteins ZO‑1 and occludin. In addition, western blot analysis demonstrated that ML7 increased the expression levels of occludin in the precipitate, but reduced its expression in the supernatant of lysed aortas. These results indicated that occludin, which is a dynamic protein at the TJ, is associated with remodeling from cell membrane to cytoplasm. The present study was the first, to the best of our knowledge, to indicate that ML7 may ameliorate VED and AS by regulating the TJ proteins ZO‑1 and occludin via mechanisms involving MLCK and MLC phosphorylation.