We reported that (23S)-25-dehydro-1-hydroxyvitamin D₃-26,23- lactone (TEI-9647) antagonizes vitamin D receptor (VDR)-mediated genomic actions of 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃] in human cells, but is agonistic in rodent cells. Human and rat VDR ligand binding domains (LBD) are similar, but differences in the C-terminal region are important for ligand binding and transactivation and might determine the agonistic/antagonistic effects of TEI-9647. We tested TEI-9647 on 1,25(OH)₂D₃ transactivation using SaOS-2 cells (human osteosarcoma) or ROS 24/1 cells (rat osteosarcoma) cotransfected with human or rodent VDR and a reporter. In both cell lines, TEI-9647 was antagonistic with wildtype (WT) human (h)VDR, but agonistic with over-expressed WT rat (r)VDR. VDR chimeras substituting the hVDR C-terminal region (AF-2 domain) with corresponding rVDR residues diminished antagonism and increased agonism of TEI-9647. However, substitution of 25 C-terminal rVDR residues with corresponding hVDR residues diminished agonism and increased antagonism of TEI-9647. Human VDR mutants (C403S, C410N) demonstrated that Cys403 and/or 410 was necessary for TEI-9647 antagonism of 1,25(OH)₂D₃ transactivation. These results suggest that species specificity of VDR, especially in the C-terminal region, determines the agonistic/antagonistic effects of TEI-9647 that determine, in part, VDR interactions with coactivators and emphasize the critical interaction between TEI-9647 and the two C-terminal hVDR Cys residues to mediate the antagonistic effect of TEI-9647.