TEI-9647 antagonizes vitamin D receptor (VDR) mediated genomic actions of 1alpha,25(OH)2D3 in human cells but is agonistic in rodent cells. The presence of Cys403, Cys410 or of both residues in the C-terminal region of human VDR (hVDR) results in antagonistic action of this compound. In the complexes of TEI-9647 with wild-type hVDR (hVDRwt) and H397F hVDR, TEI-9647 functions as an antagonist and forms a covalent adduct with hVDR according to MALDI-TOF MS. The crystal structures of complexes of TEI-9647 with rat VDR (rVDR), H305F hVDR and H305F/H397F hVDR showed that the agonistic activity of TEI-9647 is caused by a hydrogen-bond interaction with His397 or Phe397 located in helix 11. Both biological activity assays and the crystal structure of H305F hVDR complexed with TEI-9647 showed that the interaction between His305 and TEI-9647 is crucial for antagonist activity. This study indicates the following stepwise mechanism for TEI-9647 antagonism. Firstly, TEI-9647 forms hydrogen bonds to His305, which promote conformational changes in hVDR and draw Cys403 or Cys410 towards the ligand. This is followed by the formation of a 1,4-Michael addition adduct between the thiol (-SH) group of Cys403 or Cys410 and the exo-methylene group of TEI-9647.