Cytochrome P450 monooxygenase that catalyzes regio- and stereospecific hydroxylation of cholesterol and its derivatives. Hydroxylates (with R stereochemistry) the terminal methyl group of cholesterol side-chain in a three step reaction to yield at first a C26 alcohol, then a C26 aldehyde and finally a C26 acid. Regulates cholesterol homeostasis by catalyzing the conversion of excess cholesterol to bile acids via both the 'neutral' (classic) and the 'acid' (alternative) pathways. May also regulate cholesterol homeostasis via generation of active oxysterols, which act as ligands for NR1H2 and NR1H3 nuclear receptors, modulating the transcription of genes involved in lipid metabolism (By similarity). Plays a role in cholestanol metabolism in the cerebellum (PubMed:28190002). Similarly to cholesterol, hydroxylates cholestanol and may facilitate sterol diffusion through the blood-brain barrier to the systemic circulation for further degradation. Also hydroxylates retinal 7-ketocholesterol, a noxious oxysterol with pro-inflammatory and pro-apoptotic effects, and may play a role in its elimination from the retinal pigment epithelium. May play a redundant role in vitamin D biosynthesis. Catalyzes 25-hydroxylation of vitamin D3 that is required for its conversion to a functionally active form (By similarity). 5beta-cholestane-3alpha,7alpha,12alpha-triol + 5 H(+) + 3 O2 + 6 reduced [adrenodoxin] = (25R)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oate + 4 H2O + 6 oxidized [adrenodoxin] cholestanol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-26-hydroxycholestanol + H2O + 2 oxidized [adrenodoxin] (25R)-3beta-hydroxycholest-5-en-7-one-26-al + H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3beta-hydroxycholest-5-en-7-one-26-oate + H2O + 2 oxidized [adrenodoxin] (25R)-3beta,26-dihydroxycholest-5-en-7-one + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3beta-hydroxycholest-5-en-7-one-26-al + 2 H2O + 2 oxidized [adrenodoxin] 7-oxocholesterol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3beta,26-dihydroxycholest-5-en-7-one + H2O + 2 oxidized [adrenodoxin] calciol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = calcidiol + H2O + 2 oxidized [adrenodoxin] (25R)-5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-al + 2 H2O + 2 oxidized [adrenodoxin] cholesterol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-cholest-5-ene-3beta,26-diol + H2O + 2 oxidized [adrenodoxin] (25R)-3beta,4beta-dihydroxycholest-5-en-26-al + H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3beta,4beta-dihydroxycholest-5-en-26-oate + H2O + 2 oxidized [adrenodoxin] (25R)-4beta,26-dihydroxycholesterol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3beta,4beta-dihydroxycholest-5-en-26-al + 2 H2O + 2 oxidized [adrenodoxin] 4beta-hydroxycholesterol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-4beta,26-dihydroxycholesterol + H2O + 2 oxidized [adrenodoxin] (25R)-3beta-hydroxy-5-cholesten-26-al + H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3beta-hydroxy-5-cholestenoate + H2O + 2 oxidized [adrenodoxin] (25R)-cholest-5-ene-3beta,26-diol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3beta-hydroxy-5-cholesten-26-al + 2 H2O + 2 oxidized [adrenodoxin] (25R)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-al + H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-3alpha,7alpha,12alpha-trihydroxy-5beta-cholestan-26-oate + H2O + 2 oxidized [adrenodoxin] 5beta-cholestane-3alpha,7alpha,12alpha-triol + 2 H(+) + O2 + 2 reduced [adrenodoxin] = (25R)-5beta-cholestane-3alpha,7alpha,12alpha,26-tetrol + H2O + 2 oxidized [adrenodoxin] Hormone biosynthesis; cholecalciferol biosynthesis. Steroid metabolism; cholesterol degradation. Lipid metabolism; bile acid biosynthesis. Interacts with HSP70; this interaction is required for initial targeting to mitochondria. Post-translationally targeted to mitochondria. All three of the receptor proteins in the TOM complex, TOMM70, TOMM20 and TOMM22 are required for the translocation across the mitochondrial outer membrane. After translocation into the matrix, associates with the inner membrane as a membrane extrinsic protein. Expressed in the gray and white matter of cerebellum (at protein level). Acetylation of Lys-125 and Lys-285 is observed in liver mitochondria from fasted mice but not from fed mice. Mutant mice show cholestanol accumulation in the cerebellum. Belongs to the cytochrome P450 family.