The vitamin D receptor (VDR) binds to and mediates the effects of the 1,25‐dihydroxyvitamin D₃ (1,25(OH)₂D₃) hormone to alter gene transcription. A newly recognized VDR ligand is the carcinogenic bile acid, lithocholic acid (LCA). We demonstrate that, in HT‐29 colon cancer cells, both LCA and 1,25(OH)₂D₃ induce expression of cytochrome P450 3A4 (CYP3A4), an enzyme involved in cellular detoxification. We also show that LCA‐VDR stimulates transcription of gene reporter constructs containing DR3 and ER6 vitamin D responsive elements (VDREs) from the human CYP3A4 gene. Utilizing gel mobility shift, pulldown, and mammalian two‐hybrid assays, we observe that: (i) 1,25(OH)₂D₃ enhances retinoid X receptor (RXR) heterodimerization with VDR more effectively than LCA, (ii) the 1,25(OH)₂D₃‐liganded VDR‐RXR heterodimer recruits full‐length SRC‐1 coactivator, whereas this interaction is minimal with LCA unless LXXLL‐containing fragments of SRC‐1 are employed, and (iii) both 1,25(OH)₂D₃ and LCA enhance the binding of VDR to DRIP205/mediator, but unlike 1,25(OH)₂D₃‐VDR, LCA‐VDR does not interact detectably with NCoA‐62 or TRIP1/SUG1, suggesting a different pattern of LCA‐VDR comodulator association. Finally, residues in the human VDR (hVDR) ligand binding domain (LBD) were altered to create mutants unresponsive to 1,25(OH)₂D₃‐ and/or LCA‐stimulated transactivation, identifying S237 and S225/S278 as critical for 1,25(OH)₂D₃ and LCA action, respectively. Therefore, these two VDR ligands contact distinct residues in the binding pocket, perhaps generating unique receptor conformations that determine the degree of RXR and comodulator binding. We propose that VDR is a bifunctional regulator, with the 1,25(OH)₂D₃‐liganded conformation facilitating high affinity endocrine actions, and the LCA‐liganded configuration mediating local, lower affinity cellular detoxification by upregulation of CYP3A4 in the colon. J. Cell. Biochem. 94: 917–943, 2005. © 2004 Wiley‐Liss, Inc.