P450 is the collective name of a group of diversified heme protein monooxygenases constructing a large gene superfamily. Metabolic function of P450s is highly diversified as a novel term “diversozyme” has been proposed. Sterol 14-demethylase P450 (CYP51), which mediates the oxidative removal of 14α-methyl group of the sterol precursors, is an exceptional P450 that has been functionally conserved and exists in most of biological kingdoms. However, the precursors undergoing 14-demethylation are different among animals, plants and fungi in relation to their sterol biosynthetic pathways. This fact indicates that modulations of substrate specificity have occurred on CYP51 through the interaction with the precursors existing in individual kingdom. Molecular phylogenetic analysis indicated that the closest neighbor of CYP51 family is a cluster consisting of CYP7/8 families including the P450s participating in cholesterol catabolism or bile acids synthesis, CYP7A (cholesterol 7α-hydroxylase), CYP7B (oxysterol 7α-hydroxylase) and CYP8B (sterol 12α-hydroxylase). Since these P450s as well as CYP51 catalyze monooxygenation at the middle part of the steroid skeleton from the α-surface, all of them have possibly been derived from a common ancestral steroid or cyclic triterpenoid α-hydroxylase P450. CYP51, CYP7A and CYP8B are arrayed in this order in the mammalian sterol metabolic pathway. It can thus be assumed that the diversification of an ancient steroid or cyclic triterpenoid α-hydroxylase P450 through the interaction between various sterols might have played an important role in the establishment of the sterol metabolic pathway of mammals.