Background: The molecular mechanisms of exercise-induced cardioprotection are poorly understood. We recently reported that exercise training down-regulated gene expression of the Ras homolog gene family member A (RhoA). RhoA and its first effectors, the Rho-kinases (ROCK), have already been implicated in the pathogenesis of cardiovascular disease. The aim of this study was to compare the effects of a RhoA/ROCK inhibitor (fasudil) and exercise in the Apolipoprotein E knockout (ApoE-/-) mouse model of atherosclerosis. Methods: Four groups of 14 week old ApoE-/- mice were randomised as follows (n = 12/group): i) sedentary controls (Cont); ii) fasudil (Fas) treatment (100 mg/kg bodyweight/day) for 8 weeks; iii) exercise intervention (Ex:free access to running wheel for 8 weeks) and iv) exercise intervention and fasudil treatment (ExFas) for 8 weeks. Results: Phosphorylation of myosin light chain was significantly reduced in the brachiocephalic artery of all treatment groups compared with sedentary controls, implying an inhibitory effect of exercise and fasudil on the RhoA/ROCK pathway. Furthermore, atherosclerotic lesions were significantly smaller in all treatment and intervention groups compared with the control group (Fas: 34.7%, Ex: 48.3%, ExFas: 40.9% less than Control). The intima:media ratio was reduced by both exercise intervention and fasudil treatment alone or in combination (Fas: 23.6%, Ex: 35.5%, ExFas: 43.9% less than Control). Exercise alone and fasudil treatment alone also showed similar effects on plaque composition, increasing both smooth muscle cell and macrophage density. Conclusion: These results suggest that the protective effects of exercise on atherogenesis are similar to the inhibitory effects on the RhoA/ROCK signalling pathway.