Calcifediol is a prehormone which is produced by hydroxylation of vitamin D₃ (cholecalciferol) in the liver. Calcidiol is then converted in the kidneys (by the enzyme 25(OH)D-1α-hydroxylase) into calcitriol (1,25-(OH)₂D₃), a secosteroid hormone that is the active form of vitamin D. It can also be converted into 24-hydroxycalcidiol in the kidneys via 24-hydroxylation.

In medicine, blood concentration of calcidiol is considered the best indicator of vitamin D status. It is the most sensitive measure, though experts have called for improved standardization and reproducibility across different laboratories. The normal range varies widely depending on several factors, including age and geographic location. A broad reference range of 20–150 nmol/L has been suggested, while several studies have defined levels below 80 nmol/L as indicative of vitamin D deficiency.