Catalyzes the formation of 25-hydroxycholesterol from cholesterol, leading to repress cholesterol biosynthetic enzymes (PubMed:9852097). Plays a key role in cell positioning and movement in lymphoid tissues: 25-hydroxycholesterol is an intermediate in biosynthesis of 7-alpha,25-dihydroxycholesterol (7-alpha,25-OHC), an oxysterol that acts as a ligand for the G protein-coupled receptor GPR183/EBI2, a chemotactic receptor for a number of lymphoid cells (PubMed:22999953). May play an important role in regulating lipid metabolism by synthesizing a corepressor that blocks sterol regulatory element binding protein (SREBP) processing (PubMed:9852097). In testis, production of 25-hydroxycholesterol by macrophages may play a role in Leydig cell differentiation (PubMed:9852097). Interferon-stimulated gene which has broad antiviral activities against a wide range of enveloped viruses (By similarity). Catalyzes the formation of 25-hydroxycholesterol from cholesterol, leading to repress cholesterol biosynthetic enzymes (PubMed:9852097). Plays a key role in cell positioning and movement in lymphoid tissues: 25-hydroxycholesterol is an intermediate in biosynthesis of 7-alpha,25-dihydroxycholesterol (7-alpha,25-OHC), an oxysterol that acts as a ligand for the G protein-coupled receptor GPR183/EBI2, a chemotactic receptor for a number of lymphoid cells (PubMed:22999953). May play an important role in regulating lipid metabolism by synthesizing a corepressor that blocks sterol regulatory element binding protein (SREBP) processing (PubMed:9852097). As an interferon-stimulated gene, has broad antiviral activities against a wide range of enveloped viruses. Its product, 25-hydroxycholesterol, activates the ER-localized enzyme ACAT to induce internalization of accessible cholesterol on the plasma membrane and restricts virus-host membranes fusion which inhibits virus replication (By similarity). In testis, production of 25-hydroxycholesterol by macrophages plays a role in Leydig cell differentiation (By similarity). AH2 + cholesterol + O2 = 25-hydroxycholesterol + A + H2O cholesterol + H(+) + NADPH + O2 = 25-hydroxycholesterol + H2O + NADP(+) Widely expressed at low level and at higher level in the lung. Weakly expressed in the heart, lung and kidney. N-glycosylated. Mice do not display any apparent alteration in bile acid synthesis and cholesterol metabolism (PubMed:12543708). Belongs to the sterol desaturase family.