The oxysterol receptor, LXR, is a key transcriptional regulator of lipid metabolism. LXR increases expression of SREBP-1, which in turn regulates at least 32 genes involved in lipid synthesis and transport. We recently identified 25-hydroxycholesterol-3-sulfate (25HC3S) as an important regulatory molecule in the liver. We have now studied the effects of 25HC3S and its precursor, 25-hydroxycholesterol (25HC) on lipid metabolism as mediated by the LXR/SREBP-1 signaling in macrophages. Addition of 25HC3S to human THP-1 derived macrophages markedly decreased nuclear LXR protein levels. 25HC3S administration was followed by dose- and time-dependent decreases in SREBP-1 mature protein and mRNA levels. 25HC3S decreased the expression of SREBP-1 responsive genes, acetyl CoA carboxylase-1 (ACC1) and fatty acid synthase (FAS), as well as HMGR and LDLR, which are key proteins involved in lipid metabolism. Subsequently, 25HC3S decreased intracellular lipids and increased cell proliferation. In contrast to 25HC3S, 25HC acted as an LXR ligand, increasing ABCA1, ABCG1, SREBP-1, and FAS mRNA levels. In the presence of 25HC3S, 25HC and LXR agonist T0901317 stimulation of LXR targeting gene expression was repressed. We conclude that 25HC3S acts in macrophages as a cholesterol satiety signal, down-regulating cholesterol and fatty acid synthetic pathways via inhibition of LXR/SREBP signaling. A possible role of oxysterol sulfation is proposed.