Hereditary vitamin D resistant rickets (HVDRR) is an autosomal recessive disease caused by mutations in the vitamin D receptor (VDR). We studied a young Saudi Arabian girl who exhibited the typical clinical features of HVDRR but without alopecia. Analysis of her VDR gene revealed a homozygous T to C mutation in exon 7 that changed isoleucine to threonine at amino acid 268 (I268T). From crystallographic studies of the VDR ligand-binding domain, I268 directly interacts with 1,25(OH)₂D₃ and is involved in the hydrophobic stabilization of helix H12. We recreated the I268T mutation and analyzed its effects on VDR function. In ligand binding assays, the I268T mutant VDR exhibited a 5-10-fold lower affinity for [³H]1,25(OH)₂D₃ compared with the WT VDR. In transactivation assays, the I268T mutant exhibited an EC₅₀ for 1,25(OH)₂D₃ 65-fold higher compared with the WT VDR to stimulate gene transcription. Both RXR heterodimerization and coactivator binding were reduced in the I268T mutant. Analogs of 1,25(OH)₂D₃ have been proposed as potential therapeutics for patients with HVDRR. Interestingly, in protease sensitivity assays treatment with the potent vitamin D analog, 20-epi-1,25(OH)₂D₃ stabilized I268T mutant proteolytic fragments better than 1,25(OH)₂D₃. Moreover, 20-epi-1,25(OH)₂D₃ restored transactivation of the I268T mutant to levels exhibited by WT VDR treated with 1,25(OH)₂D₃. In conclusion, we describe a novel mutation I268T in the VDR LBD that alters ligand binding, RXR heterodimerization, and coactivator binding. These combined defects in VDR function cause resistance to 1,25(OH)₂D₃ action and result in the syndrome of HVDRR.