Membrane damage during traumatic brain injury (TBI) alters the brain homeostasis of cholesterol and other lipids. Cholesterol 24S-hydroxylase (Cyp46) is a cholesterol metabolic enzyme that is increased after TBI. Here, we systematically examined the effects of the enzymatic product of Cyp46, 24S-hydroxycholesterol, on the cholesterol regulatory genes, SREBP-1 and 2, their posttranslational regulation, and their effects on gene transcription. 24S-hydroxycholesterol increased levels of SREBP-1 mRNA and full-length protein but did not change levels of cleaved SREBP-1, consistent with the role of 24-hydroxycholesterol as an LXR agonist. In contrast, 24S-hydroxycholesterol decreased levels of LXR-independent SREBP-2 mRNA, full-length protein, and SREBP-2 active cleavage product. We examined the downstream effects of changes to these lipid regulatory factors by studying cholesterol and fatty acid synthesis genes. In neuroblastoma cells, 24S-hydroxycholesterol decreased mRNA levels of the cholesterol synthesis genes HMG CoA reductase, squalene synthase, and FPP synthase but did not alter levels of the mRNA of fatty acid synthesis genes acetyl CoA carboxylase or fatty acid synthase. After TBI, as after 24S-hydroxycholesterol treatment in vitro, SREBP-1 mRNA levels were increased while SREBP-2 mRNA levels were decreased. Also similar to the in vitro results with 24S-hydroxycholesterol, HMG CoA reductase and squalene synthase mRNA levels were significantly decreased. Fatty acid synthase mRNA levels were not altered but acetyl CoA carboxylase mRNA levels were significantly decreased. Thus, changes to transcription of cholesterol synthesis genes after TBI were consistent with increases in Cyp46 activity, but changes to fatty acid synthesis genes must be regulated by other mechanisms.