Rho kinases have been shown to be involved in the pathogenesis of atherosclerosis. This study examined the effects of fasudil, a specific Rho kinase inhibitor, on plaque development and progression in atherosclerotic mice. Sixty apolipoprotein E-knockout (apoE-KO) mice were fed a high-fat diet. Mice started to receive fasudil at the same time as fat feeding (early treatment), or after 12 weeks of fat feeding (delayed treatment). In each administrative schedule, mice were divided into three groups: low dose fasudil group (30 mg/kg/day), high dose fasudil group (100mg/kg/day) and control group (tap water) (n=10, respectively). Plaque size was determined by using ultrasound biomicroscopy (UBM) and histological examinations. Brachiocephalic artery UBM analysis showed that in early treatment, both doses of fasudil significantly reduced lesion size compared with the controls (P<0.05). In delayed-fasudil treatment, plaque area was reduced by 54% (P<0.05) after 12 weeks of treatment at a high dose of fasudil (100mg/kg/day). The UBM findings were confirmed by histological studies at the corresponding arterial sites. The beneficial effect was also observed in the left common carotid arteries that delayed-fasudil treatment reduced the plaque size in a dose-dependent manner. The arterial intima-medial thickness (IMT) and maximal flow velocity of both arteries were lower in fasudil-treated group (100mg/kg/day) in comparison with the control mice. Furthermore, fasudil treatment (100mg/kg/day) reduced the macrophage accumulation in atherosclerotic lesions. However, fasudil had no effects on blood pressure and plasma lipid concentrations in both studies. In conclusion, our studies showed that blocking Rho kinase reduced both the early development and later progression of atherosclerotic plaques in apoE-KO mice by using a novel micro-ultrasound approach.