The nuclear receptors liver X receptor (LXR) LXRα and LXRβ are differentially expressed ligand-activated transcription factors that induce genes controlling cholesterol homeostasis and lipogenesis. Synthetic ligands for both receptor subtypes activate ATP binding cassette transporter A1 (ABCA1)-mediated cholesterol metabolism, increase reverse cholesterol transport, and provide atheroprotection in mice. However, these ligands may also increase hepatic triglyceride (TG) synthesis via a sterol response element binding protein 1c (SREBP-1c)-dependent mechanism through a process reportedly regulated by LXRα. We studied pan-LXRα/β agonists in LXRα knockout mice to assess the contribution of LXRβ to the regulation of selected target genes. In vitro dose-response studies with macrophages from LXRα-/- and β-/- mice confirm an equivalent role for LXRα and LXRβ in the regulation of ABCA1 and SREBP-1c gene expression. Cholesterol-efflux studies verify that LXRβ can drive apoA1-dependent cholesterol mobilization from macrophages. The in vivo role of LXRβ in liver was further evaluated by treating LXRα-/- mice with a pan-LXRα/β agonist. High-density lipoprotein (HDL) cholesterol increased without significant changes in plasma TG or very low density lipoprotein. Analysis of hepatic gene expression consistently revealed less activation of ABCA1 and SREBP-1c genes in the liver of LXRα null animals than in treated wild-type controls. In addition, hepatic CYP7A1 and several genes involved in fatty acid/TG biosynthesis were not induced. In peripheral tissues from these LXRα-null mice, LXRβ activation increases ABCA1 and SREBP-1c gene expression in a parallel manner. However, putative elevation of SREBP-1c activity in these tissues did not cause hypertriglyceridemia. In summary, selective LXRβ activation is expected to stimulate ABCA1 gene expression in macrophages, contribute to favorable HDL increases, but circumvent hepatic LXRα-dominated lipogenesis.