Low levels of 25-hydroxy vitamin D (25(OH)D) are associated with cardiovascular diseases. Herein, we tested the hypothesis that vitamin D deficiency could be a causal factor in atherosclerotic vascular changes and vascular calcification. Aortic root sections of vitamin D receptor knockout (VDR(-/-)) mice that were stained for vascular calcification and immunostained for osteoblastic differentiation factors showed more calcified areas and a higher expression of the osteogenic key factors Msx2, Bmp2, and Runx2 than the wild-type mice (P<0.01). Data from LDL receptor knockout (LDLR(-/-)) mice that were fed western diet with either low (50 IU/kg), recommended (1,000 IU/kg), or high (10,000 IU/kg) amounts of vitamin D(3) over 16 weeks revealed increasing plasma concentrations of 25(OH)D (P<0.001) with increasing intake of vitamin D, whereas levels of calcium and phosphorus in plasma and femur were not influenced by the dietary treatment. Mice treated with the low vitamin D diet had more calcified lesions and a higher expression of Msx2, Bmp2, and Runx2 in aortic roots than mice fed recommended or high amounts of vitamin D (P<0.001). Taken together, these findings indicate vitamin D deficiency as a risk factor for aortic valve and aortic vessel calcification and a stimulator of osteogenic key factor expression in these vascular areas.