Liver X Receptors (LXR α/β), sensors of cholesterol, are involved in regulation of lipid metabolism, and are promising drug targets against many diseases, including atherosclerosis, metabolic syndromes, and cancers. Activation of LXRα can cause adverse effects, and a current focus is a search for LXRβ-selective agonists. An understanding of the ligand selective mechanisms is critical in the design and identification of LXRβ-selective ligands because the α and β isoforms, LXRα-Val263 and LXRβ-Ile277 have only minor structural differences in the ligand binding domain (LBD). In this work, in silico and in vitro studies have been performed. For the first time, we report that the ligand LXRβ-selectivity relies on structural differences between LXRα-Val263 and LXRβ-Ile277, and also on conformational changes at Leu274 and Ala275. These residues are the same in both α and β isoforms, but their conformational changes when interacting with selective ligands are different. Mutation studies indicate that replacing both Val263 and Ile277 with alanine residues does not completely nullify the β-selectivity. High β-binding selectivity can be related to the synergistic effects of other residues that interact with the ligand in the LBD although these residues are identical in both LXRα and LXRβ. This work offers a path to the design and optimization of selective LXRβ agonists.