Although it has been well documented that vitamin D receptor (VDR) activation influences the expression of various genes involved in calcium homeostasis and cell differentiation, the physiological role of VDR action in hemostasis remains unclear. We studied thrombogenicity in normocalcemic VDR knock-out (KO) mice on a high calcium diet in comparison with that in wild-type mice and that in hypocalcemic VDRKO mice fed a regular diet. Platelet aggregation was significantly enhanced in normocalcemic VDRKO mice. Aortic endothelial nitric-oxide (NO) synthase expression and urinary NOx excretion were reduced in hypocalcemic VDRKO mice but not in normocalcemic VDRKO mice. The gene expression of antithrombin in the liver and that of thrombomodulin in the aorta, liver and kidney were down-regulated in hypo- and normocalcemic VDRKO mice, whereas tissue factor gene expression in the liver and kidney was up-regulated in VDRKO mice regardless of plasma calcium level. Furthermore, VDRKO mice manifested an exacerbated multi-organ thrombus formation after exogenous lipopolysaccharide injection regardless of the calcemic conditions. These results demonstrate that the vitamin D-VDR system plays a pivotal role in antithrombogenicity in vivo.