Vitamin D receptor (VDR) antagonists prevent the VDR activation function helix 12 from folding into its active conformation, thus affecting coactivator recruitment and antagonizing the transcriptional regulation induced by 1α,25-dihydroxyvitamin D3. Here, we report the crystal structure of the zebrafish VDR ligand-binding domain in complex with the ZK168281 antagonist, revealing that the ligand prevents optimal folding of the C-terminal region of VDR. This interference was confirmed by hydrogen−deuterium exchange mass spectrometry (HDX-MS) in solution.