It was previously shown that CYP3A4 is induced in the human intestinal Caco-2 cell model by treatment with 1α,25-dihydroxy vitamin D₃ (1,25-D₃). We demonstrate the vitamin D analog, 19-nor-1α,25-dihydroxy vitamin D₂, is also an effective inducer of CYP3A4 in Caco-2 cells, but with half the potency of 1,25-D₃. We report that treatment of LS180 cells, a human intestinal cell line, with 1 to 10 nM 1,25-D₃ dose dependently increased CYP3A4 protein and CYP3A4 mRNA expression.CYP3A4- and CYP3A23-promoter-Luciferase reporter constructs transiently transfected into LS180 cells were transcriptionally activated in a dose-dependent manner by 1,25-D₃, whereas mutation of the nuclear hormone receptor binding motif (ER6) in the CYP3A4 promoter abrogated 1,25-D₃ activation of CYP3A4. Although theCYP3A4 ER6 promoter element has been shown to bind the pregnane X receptor (PXR), this receptor does not mediate 1,25-D₃ induction of CYP3A4 because a) PXR is not expressed in Caco-2 cells; b) PXR mRNA expression is not induced by 1,25-D₃ treatment of LS180 cells; and c) the ligand binding domain of human PXR was not activated by 1,25-D₃. 1,25-D₃ uses the vitamin D receptor to induce CYP3A4 because a) the vitamin D receptor (VDR)-retinoid X receptor (RXR) heterodimer binds specifically to the CYP3A4 ER6; b) selective mutation of the CYP3A4 ER6 disrupted the binding of VDR-RXR; and c) reporter constructs containing only three copies of the CYP3A4 ER6 linked to a TK-CAT reporter were activated by 1,25-D₃ only in cells cotransfected with a human VDR expression plasmid. These data support the hypothesis that 1,25-D₃ and VDR induce expression of intestinal CYP3A by binding of the activated VDR-RXR heterodimer to theCYP3A PXR response element and promoting gene transcription.