Vitamin D insufficiency is highly associated with cardiovascular morbidity and mortality. We have demonstrated enhanced vascular calcification in LDL receptor knockout (LDLR−/−) mice fed a diet low in vitamin D. This study aimed to investigate the impact of a diet low in vitamin D on vascular calcification in wild-type (WT) mice lacking atherosclerotic plaques and the effects of a persistent and discontinuous vitamin D insufficiency on atherosclerotic plaque composition in LDLR−/− mice. The study was performed with 4-wk-old male WT and LDLR−/− mice that were fed a normal calcium/phosphate Western diet (210 g/kg fat, 1.5 g/kg cholesterol) containing either adequate (+D; 1000 IU/kg) or low (−D; 50 IU/kg) amounts of vitamin D-3 for 16 wk. Four groups of LDLR−/− mice received 1 of the 2 diets for additional 16 wk (total 32 wk) and were compared with mice fed the diets for only 16 wk. WT and LDLR−/− mice that were fed the −D diet for 16 wk tended to develop more calcified spots in the aortic valve than mice fed the +D diet (+50% and +56%, respectively; P < 0.10). In LDLR−/− mice, the extent of calcification increased from week 16 to week 32 and was higher in the −D than in the +D group (P < 0.05). The calcification, owing to the −D diet, was accompanied by highly expressed osteoblast differentiation factors, indicating a transdifferentiation of vascular cells to osteoblast-like cells. Feeding the +D diet subsequent to the −D diet reduced the vascular calcification (P < 0.05). LDLR−/− mice fed the −D diet for 32 wk had higher plaque lipid depositions (+48%, P < 0.05) and a higher expression of cluster of differentiation 68 (+31%, P < 0.05) and tumor necrosis factor α (+134%, P < 0.001) than the +D group. Collectively, the findings imply low vitamin D status as a causal factor for vascular calcification and atherosclerosis. J. Nutr. 144: 638-646, 2014.