Vitamin D is a prohormone that undergoes biological metabolism to become metabolically active and is available in two isoforms: ergocalciferol (vitamin D2) and cholecalciferol (vitamin D3). Ergocalciferol is mainly derived from fungi, plants, and yeast, whereas cholecalciferol is found in the epidermis of vertebrates. The metabolism of vitamin D starts with exposure to sunlight (or UV light in the range of 290–320 nm), which induces a rearrangement of 7-dehydrocholesterol into vitamin D3. It is then bound by vitamin D-binding protein for further metabolism in the liver and kidneys by many cytochrome P450 (CYP enzymes). In the liver, it is hydroxylated into 25-hydroxycholecalciferol (calcidiol) and is then hydroxylated in the kidney into 1,25-dihydroxycholecalciferol (calcitriol), which is considered the metabolically active form of vitamin D. Both calcidiol and calcitriol are tightly regulated by CYP24A1, a mitochondrial inner membrane cytochrome P50 enzyme. This is the enzyme responsible for the inactivation of vitamin D, which is then excreted via the biliary tract. Vitamin D is also regulated by parathyroid hormone (PTH) (in response to low calcium levels) and fibroblast growth factor-23 (FGF-23), mainly via changes in the expression levels of CYP 50 enzymes involved in the metabolism of vitamin D. The molecular effects of vitamin D are wide and directly modulate cyclooxygenase-2 (COX-2), the P38 MAPK pathway, and NFkB, among others. These molecular effects are thought to contribute to its immunomodulatory and anti-inflammatory roles, thus giving rise to its antineoplastic analogs and their antiproliferative role within cancer treatment, which are discussed closely herein.