This chapter reviews the role of 25-hydroxyvitamin D 24-hydroxylase in vitamin D metabolism. The 24-hydroxylase enzyme is a widely distributed component of the vitamin D pathway. It is regulated by a spectrum of hormones and functions to synthesise 24-hydroxylated metabolites, with preferential action in promoting bone mineralization and possibly other undisclosed cellular functions that may become evident through the use of gene knockout models. It also plays a central role in directing the metabolic turnover of several 25-hydroxylated vitamin D metabolites