In humans, the brain represents only about 2% of the body's mass but contains about one-quarter of the body's free cholesterol. Cholesterol is synthesized de novo in brain and removed by metabolism to oxysterols. 24S-Hydoxycholesterol represents the major metabolic product of cholesterol in brain, being formed via the cytochrome P450 (CYP) enzyme CYP46A1. CYP46A1 is expressed exclusively in brain, normally by neurons. In this study, we investigated the effect of 24S-hydroxycholesterol on the proteome of rat cortical neurons. With the use of two-dimensional liquid chromatography linked to nanoelectrospray tandem mass spectrometry, over 1040 proteins were identified including members of the cholesterol, isoprenoid and fatty acid synthesis pathways. With the use of stable isotope labeling technology, the protein expression patterns of enzymes in these pathways were investigated. 24S-Hydroxycholesterol was found to down-regulate the expression of members of the cholesterol/isoprenoid synthesis pathways including 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 1 (EC 18.104.22.168), diphosphomevalonate decarboxylase (EC 22.214.171.124), isopentenyl-diphosphate delta isomerase (EC 126.96.36.199), farnesyl-diphosphate synthase (Geranyl trans transferase, EC 188.8.131.52), and dedicated sterol synthesis enzymes, farnesyl-diphosphate farnesyltransferase 1 (squalene synthase, EC 184.108.40.206) and methylsterol monooxygenase (EC 220.127.116.11). The expression of many enzymes in the cholesterol/isoprenoid and fatty acid synthesis pathways are regulated by the membrane-bound transcription factors named sterol regulatory element-binding proteins (SREBPs), which themselves are both transcriptionally and post-transcriptionally regulated. The current proteomic data indicates that 24S-hydroxycholesterol down-regulates cholesterol synthesis in neurons, possibly, in a post-transcriptional manner through SREBP-2. In contrast to cholesterol metabolism, enzymes responsible for the synthesis of fatty acids were not found to be down-regulated in neurons treated with 24S-hydroxycholesterol, while apolipoprotein E (apo E), a cholesterol trafficking protein, was found to be up-regulated. Taken together, this data leads to the hypothesis that, in times of cholesterol excess, 24S-hydroxycholesterols signals down-regulation of cholesterol synthesis enzymes through SREBP-2, but up-regulates apo E synthesis (through the liver X receptor) leading to cholesterol storage and restoration of cholesterol balance.