Lithocholic acid (2) was identified as a second endogenous ligand of vitamin D receptor (VDR), though its activity is very weak. In this study, we designed novel lithocholic acid derivatives based on the crystal structure of VDR-ligand-binding domain (LBD) bound to 2. Among the synthesized compounds, 6 bearing a 2-hydroxy-2-methylprop-1-yl group instead of the 3-hydroxy group at the 3α-position of 2 showed dramatically increased activity in HL-60 cell differentiation assay, being at least 10 000 times more potent than lithocholic acid (2) and 3 times more potent than 1α,25-dihydroxyvitamin D3 (1). Although the binding affinities of 6 and its epimer 7 were less than that of 1, their transactivation activities were greater than that of 1. X-ray structure analyses of VDR LBD bound to 6 or 7 showed that the binding positions of these compounds in the ligand-binding pocket are similar to that of 1.