Vitamin D3 plays a central role in calcium and phosphate homeostasis and is essential for the proper development and maintenance of bone. To exert its biological activities, vitamin D3 has to receive enzymatic transformation to the active form, 1,25-dihydroxyvitamin D3. The first step is the 25-hydroxylation reaction in the liver that produces 25-hydroxyvitamin D3, the major circulating form of vitamin D3. The 25-hydroxylation reaction is the prerequisite step for the subsequent 1 alpha-hydroxylation and 24-hydroxylation reactions in the kidney. The 1 alpha-hydroxylation reaction produces the active form of vitamin D3, whereas 24-hydroxylation reaction leads to inactivation. Both reactions are strictly controlled by parathyroid hormone, 1,25-dihydroxyvitamin D3, and calcium in a reciprocal manner in the kidney. At present, six cytochrome P450s (CYP2C11, 27A1, 2D25, 2R1, 3A4, and 2J3) are found to exhibit vitamin D 25-hydroxylation activities, and CYP27B1 and CYP24 are proved to be 1 alpha-hydroxylase and 24-hydroxylase, respectively. The main focus of this review is to summarize the properties of individual P450 in light of their catalytic activities to understand their physiological significance.