This study investigates the role of the ligand-activated transcription factor AHR in repopulating the intestinal lining. Using organoid-derived cells and decellularized mouse intestinal scaffolds to investigate the importance of AHR in regulating intestinal regeneration, we found that silencing AHR expression hinders the capacity of colonic cells to repopulate decellularized colons. We therefore propose that AHR may play an important role in regulating intestinal regeneration. The ligand-dependent nature of AHR activity may provide an opportunity to interfere with disorders such as cancer and inflammatory bowel diseases which are caused by dysregulation in intestinal tissue renewal.