Atherosclerosis, the leading cause of cardiovascular disease responsible for the majority of deaths worldwide, cannot be sufficiently explained by established risk factors, including hypercholesterolemia. Elevated plasma homocysteine is an independent risk factor for atherosclerosis and is strongly linked to cardiovascular mortality. However, the role of homocysteine in atherosclerosis is still insufficiently understood. Previous research in this area has been also hampered by the lack of reproducible in vivo models of atherosclerosis that resemble the human situation. Here, we have developed and applied an automated system for vessel wall injury that leads to more homogenous damage and more pronounced atherosclerotic plaque development, even at low balloon pressure. Our automated system helped to glean vital details of cholesterol-independent changes in the aortic wall of balloon-injured rabbits. We show that deficiency of B vitamins, which are required for homocysteine degradation, leads to atherogenic transformation of the aorta resulting in accumulation of macrophages and lipids, impairment of its biomechanical properties and disorganization of aortic collagen/elastin in the absence of hypercholesterolemia. A combination of B vitamin deficiency and hypercholesterolemia leads to thickening of the aorta, decreased aortic water diffusion, increased LDL-cholesterol and impaired vascular reactivity compared to any single condition. Our findings suggest that deficiency of B vitamins leads to atherogenic transformation of the aorta even in the absence of hypercholesterolemia and aggravates atherosclerosis development in its presence.