Abstract Hydroxylated chalcones are phytochemicals which are biosynthetic precursors of flavonoids and their 1,3-diaryl-prop-2-en-1-one structure is used as a scaffold for drug development. In this study, the structure-dependent activation of aryl hydrocarbon receptor (AhR)-responsive CYP1A1, CYP1B1, and UGT1A1 genes was investigated in Caco2 colon cancer cells and in non-transformed young adult mouse colonocytes (YAMC) cells. The effects of a series of di- and trihydroxychalcones as AhR agonists was structure dependent with maximal induction of CYP1A1, CYP1B1, and UGT1A1 in Caco2 cells observed for compounds containing 2,2′-dihydroxy substituents and this included 2,2′-dihydroxy-, 2,2′,4′-trihydroxy-, and 2,2′,5′-trihydroxychalcones. In contrast, 2′,4,5′-, 2′3′,4′-, 2′,4,4′-trihydroxy, and 2′,3-, 2′,4-, 2′,4′-, and 2′,5-dihydroxychalcones exhibited low to non-detectable AhR activity in Caco2 cells. In addition, all of the hydroxychalcones exhibited minimal to non-detectable activity in YAMC cells, whereas 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induced CYP1A1, CYP1B1, and UGT1A1 in Caco2 and YAMC cells. The activity of AhR-active chalcones was confirmed by determining their effects in AhR-deficient Caco2 cells. In addition, 2,2′-dihydroxychalcone induced CYP1A1 protein and formation of an AhR-DNA complex in an in vitro assay. Simulation and modeling studies of hydroxylated chalcones confirmed their interactions with the AhR ligand-binding domain and were consistent with their structure-dependent activity as AhR ligands. Thus, this study identifies hydroxylated chalcones as AhR agonists with potential for these phytochemicals to impact AhR-mediated colonic pathways.