Examination of klotho-deficient (kl/kl) mice showed that they had developed vascular medial calcification. Von Kossa and van Gieson staining showed calcification of the straight elongated elastic lamellae. Interestingly, elastic fibers, but not the abundant collagen fibrils surrounding them, were preferentially calcified in the kl/kl aorta. Unlike normal vascular smooth muscle cells (VSMCs), tunica media cells of kl/kl mice had developed cisterns of rough endoplasmic reticulum and Golgi apparatus; thus, they resembled matrix-synthesizing cells rather than authentic VSMCs. Additionally, many matrix vesicle-like structures and calcifying nodules were observed in the vicinity of these VSMC-like cells in kl/kl tunica media. Several calcifying nodules made contact with the amorphous organic materials, allowing the extension of calcification into the amorphous materials from these contact points. In contrast, mineral deposition within the amorphous organic materials was also observed, indicating dystrophic calcification. These observations suggest 2 possible pathways of vascular calcification: one involving matrix vesicle-like structures that could include mineral crystals and the other involving the deposition of calcium phosphates within the amorphous materials. The former pathway suggests the possibility of trans-differentiation of VSMCs into the osteoblastic lineage that could induce biological calcification of vascular tissues, in addition to the classical hypothesis involving dystrophic calcification. Here, we review our current findings on the process of vascular medial calcification of the aorta in klotho-deficient mice.