AbstractCholesterol serves as a building material for cellular membranes and plays an important role in cellular metabolism. The brain relies on its own cholesterol biosynthesis, which starts during embryonic development. Cholesterol is synthesized from two immediate precursors, desmosterol and 7‐dehydrocholesterol (7‐DHC). Mutations in the DHCR24 enzyme, which converts desmosterol into cholesterol, lead to desmosterolosis, an autosomal recessive developmental disorder. In this study, we assessed the brain content of desmosterol, 7‐DHC, and cholesterol from development to adulthood, and analyzed the biochemical, molecular, and anatomical consequences ofDhcr24mutations on the sterol profile in a mouse model of desmosterolosis and heterozygousDhcr24+/−carriers. Our HPLC‐MS/MS studies revealed that by P0 desmosterol almost entirely replaced cholesterol in theDhcr24‐KO brain. The greatly elevated desmosterol levels were also present in theDhcr24‐Het brains irrespective of maternal genotype, persisting into adulthood. Furthermore,Dhcr24‐KO mice brains showed complex changes in expression of lipid and sterol transcripts, nuclear receptors, and synaptic plasticity transcripts. CulturedDhcr24‐KO neurons showed increased arborization, which was also present in theDhcr24‐KO mouse brains. Finally, we observed a shared pathophysiological mechanism between the mouse models of desmosterolosis and Smith–Lemli–Opitz syndrome (a genetic disorder of conversion of 7‐DHC to cholesterol).