Context <p>Various vitamin D₃ (VD3) derivatives have been developed as potent inhibitors against vitamin D receptor (VDR) for blocking the specific binding of active vitamin D to VDR. Among them, some fluorinated VD3 derivatives were revealed to possess an improved binding affinity to VDR. However, the reason for this significant improvement has not been elucidated. In the present study, we investigated the specific interactions between VDR and the fluorinated VD3 derivatives using molecular mechanics and ab initio fragment molecular orbital (FMO) calculations. Additionally, we considered the diastereomers based on both C3- and C13-stereocenters of these VD3 derivatives and investigated their interactions with VDR, elucidating that the evaluated binding energies between VDR and the diastereomers are comparable to the trend of their binding affinities to VDR obtained by the previous experiment. Based on the FMO results, the effect of fluorination of VD3 derivatives on their specific interactions with VDR was also elucidated at atomic and electronic levels. The present finding may offer valuable insights for proposing novel inhibitors targeting VDR.</p> Methods <p>The structures of our target VD3 derivatives were optimized using the B3LYP/6-31G(d,p) method of Gaussian 16 (G16). The charge distributions of the optimized structures were calculated by constrained electrostatic potential (RESP) analysis using the HF/6-31G(d) method of G16. The RESP charges were used for describing the electrostatic interactions between each derivative and the VDR residues in classical molecular mechanics (MM) and molecular dynamics (MD) calculations. As the initial structure of VDR, the X-ray crystal structure (PDB ID: 1DB1) was used, and by using the fitting tool (gmx confrms) of the MD simulation program GROMACS, the initial structures of the VDR − derivative complexes were created. To obtain their stable structures, the classical MM method of AMBER18 was used. The tleap command of AMBER18 was employed for adding hydrogen and fluorine atoms to the PDB structure and generating solvation water molecules within 8&#xa0;Å around the complex. This solvated structure of the complex was optimized using the MM method by considering water molecules explicitly. In the MM optimizations, the AMBER14SB force field, the generalized AMBER force field, and the TIP3P model were assigned for VDR, derivatives, and water molecules, respectively. We furthermore conducted MD simulations (100&#xa0;ns at 300&#xa0;K) for the MM optimized structures using the same force fields and confirmed the stability of the complex. Finally, to elucidate the specific interactions between VDR and each VD3 derivative at an electronic level, we investigated the electronic properties of the complexes in explicit water using the ab initio fragment molecular orbital (FMO) method. We employed the ab initio MP2/6-31G(d) method of the FMO calculation program ABINIT-MP Ver6.0, to accurately investigate the π–π stacking, NH–π, and CH–π interactions as well as the hydrogen-bonding and electrostatic interactions between the VDR residues and the derivatives. Furthermore, to highlight the critical VDR residues for the binding between VDR and the derivative, we investigated the inter-fragment interaction energies obtained by the FMO calculations.</p>