To this day, improvements in colon cancer patient survival remain strongly undermined by the high rates of tumour recurrence. Patterns of therapeutic failure seen in patients are consistent with a steady accumulation of drug-resistant cancer cells exhibiting functional characteristics of cancer stem cell (CSC)/tumour-initiating cells (TIC). Here we report that the nuclear receptor PXR/NR1I2, previously shown to enhance overall chemoresistance of colon and other solid tumours, is a master-regulator of colon TIC treatment resistance and of their ability to generate post-treatment tumour relapse. We first observed that expression levels of PXR showed a significant positive correlation with those of the TIC and chemoresistance marker ALDH1A1 in primary and metastasis CRC samples, and that a molecular signature associated with poor progression-free survival of CRC patients (Oh SC. et al, Gut 61(9):1291-1298, 2012) was strongly enriched in PXR-overexpressing CRC cells. The enrichment of PXR paralleled that of TIC markers upon treatment of several human colorectal cancer cell lines and of cells isolated from human CRC biopsies with standard-of-care chemotherapy. PXR was strongly enriched in colorectal cancer cells displaying self-renewal abilities and its expression was essential for the enrichment of cells expressing TIC markers (eg. ALDH1A1, ABCG2, POU5F1, LGR5…) following in vitro and in vivo exposure to cytotoxic agents, suggesting that PXR-expressing cells are better equipped to resist chemotherapy. PXR was found to drive the expression of multiple genes involved in therapy resistance (drug transporters, metabolizing enzymes, anti-apoptotic genes, TERT…) in enriched ALDH-positive colon TICs. Finally, PXR down-regulation altered the self-renewal of colon TICs in vitro and hampered their capacity to resist chemotherapy in vivo, leading to significant delays of post-chemotherapy tumour relapse and secondary xenograft growth. This study strongly suggests that targeting PXR may represent a novel treatment strategy to prevent drug resistance and recurrence through the sensitization of TICs to standard chemotherapy.