The nuclear vitamin D receptor (VDR) can recognize and bind the 1,25(OH)2D3 (1,25D) hormone and the carcinogenic bile acid lithocholic acid (LCA) as cognate ligands. VDR liganded with 1,25D or LCA can elicit expression of the metabolizing enzyme CYP3A4 within enteric cells, suggesting an important role for VDR in cellular detoxification in addition to its well-defined endocrine effects on calcium and phosphate homeostasis. Using a range of biochemical and cell-based hybrid interaction approaches, we find that binding of LCA by VDR appears to result in a distinct pattern of co-modulator recruitment compared to that directed by 1,25D, implying that VDR may possess the capacity to differentially sense and respond to each of its cognate ligands. To further explore this concept, we investigated how alternately liganded VDR can impact upon the selective expression of genes that comprise the enteric detoxification gene network and we observe that expression of the multi-drug resistance-associated protein 2 (MRP2) in Caco-2 cells is preferentially enhanced by VDR when liganded to the secondary bile acid derivative, 3-keto LCA over its high-affinity 1,25D ligand. Collectively these data implicate VDR as a dual-functional mediator of bile acid-derived and endocrine-based signals. In addition, we have also examined if VDR, through the targeted expression of metabolizing-related genes, has the capacity to contribute to the localised metabolism of steroid-based hormones in tissues such as prostate. In prostate cells we observe significant enhancement of CYP3A4, CYP3A5 and CYP19 (aromatase) expression through 1,25-VDR, an effect that becomes progressively silenced when examined in the context of androgen-insensitive prostate cancer cells. Based on these data we propose a novel role for 1,25-VDR in prostate through limiting the intracellular bioavailability of testosterone to be metabolized to dihydrotestosteone (DHT), the principal androgenic hormone driving growth of prostate epithelium.