The vitamin D receptor (VDR) mediates the physiological and pharmacological actions of 1α,25-dihydroxyvitamin D(3) in bone and calcium metabolism, cellular growth and differentiation, and immunity. VDR also responds to secondary bile acids and belongs to the NR1I subfamily of the nuclear receptor superfamily, which regulates expression of xenobiotic metabolism genes. When compared to knockout mouse investigations of the other NR1I nuclear receptors, pregnane X receptor and constitutive androstane receptor, an understanding of the role of VDR in xenobiotic metabolism remains limited. We examined the effect of VDR deletion in a mouse model of cholestasis. We performed bile duct ligation (BDL) on VDR-null mice and compared blood biochemistry, mRNA expression of genes involved in bile acid and bilirubin metabolism, cytokine production, and expression of inflammatory regulators with those of wild-type mice. VDR-null mice had elevated plasma conjugated bilirubin levels three days after BDL compared with wild-type mice. Urine bilirubin levels and renal mRNA and/or protein expression of multidrug resistance-associated proteins 2 and 4 were decreased in VDR-null mice, suggesting impaired excretion of conjugated bilirubin into urine. While VDR-null kidney showed mRNA expression of interleukin-6 (IL-6) after BDL and VDR-null macrophages had higher IL-6 protein levels after lipopolysaccharide stimulation, the induction of intestinal Il6 mRNA expression and plasma IL-6 protein levels after BDL was impaired in VDR-null mice. Immunoblotting analysis showed that expression of an immune regulator, IκBα, was elevated in the jejunum of VDR-null mice, a possible mechanism for the attenuated induction of Il6 expression in the intestine after BDL. Increased expression of IκBα may be a consequence of compensatory mechanisms for VDR deletion. These results reveal a role of VDR in bilirubin clearance during cholestasis. VDR is also suggested to contribute to tissue-selective immune regulation.