The orphan nuclear receptor pregnane X receptor (PXR) plays an important role in the detoxification of foreign and endogenous chemicals, including bile acids. PXR promotes bile acid elimination by activating bile acid detoxifying enzymes and transporters. Certain bile acids are known to promote colonic carcinogenesis by inducing colon cancer cell apoptosis. However, whether and how PXR plays a role in colon cancer apoptosis has not been reported. In this study, we showed that activation of PXR by genetic (using a constitutively activated PXR) or pharmacological (using PXR agonist rifampicin) means protected the PXR-overexpressing colon cancer HCT116 cells from deoxycholic acid (DCA)-induced apoptosis. Interestingly, activation of PXR also protected HCT116 cells from adriamycin-induced cell death, suggesting that the anti-apoptotic effect of PXR was not bile acid-specific. Moreover, the anti-apoptotic effect of PXR in HCT116 cells appeared to be independent of xenobiotic enzyme regulation, since these cells had little basal and inducible expression of bile acid detoxifying enzymes. Instead, SuperArray analysis showed that PXR-mediated DCA resistance was associated with up-regulation of multiple anti-apoptotic genes, including BAG3, BIRC2 and MCL-1, and down-regulation of pro-apoptotic genes, such as BAK1 and TP53/p53. Treatment with RIF in colon cancer LS180 cells, a cell line known to express endogenous PXR, also inhibited apoptosis. Activation of PXR in transgenic mice inhibited bile acid-induced colonic epithelial apoptosis and sensitized mice to dimethylhydrazine (DMH)-induced colonic carcinogenesis, suggesting that the anti-apoptotic effect of PXR is conserved in normal colon epithelium. In summary, our results have established the anti-apoptotic role of PXR in both human colon cancer cells and normal mouse colon epithelium.