Chalcones are phenolic compounds that can be isolated from plants. Previous studies have described some pharmacological applications for these compounds. Making use of our established reporter gene system, we determined the effect of five hydroxychalcones—2-hydroxychalcone, 2′-hydroxychalcone, 4-hydroxychalcone, 4,2′,4′-trihydroxychalcone, and 3,4,2′,4′-tetrahydroxychalcone—on the cellular xenobiotic responsive element (XRE)-transactivation. The interference of chalcones acting against polycyclic aromatic hydrocarbon (PAH)–DNA binding was also examined. Enzyme inhibition assays of cytochrome P450 (CYP) 1A1 and CYP1B1 were initially performed on recombinant protein expressed in insect microsomes. 2′-Hydroxychalcone and 2-hydroxychalcone were the most effective among the tested hydroxychalcones. The two hydroxychalcones had comparable IC 50 values for CYP1A1 and CYP1B1, which were determined to be at the micromolar and submicromolar range, respectively. However, reporter gene assays indicated that 2′-hydroxychalcone suppressed XRE-transactivation, whereas 2-hydroxychalcone induced it when 7,12-dimethylbenz[a]anthracene (DMBA) was co-administered. In the absence of DMBA, 10μM 2-hydroxychalcone and 2′-hydroxychalcone increased XRE-transactivation by 18- and 2.5-fold, respectively, while other chalcones did not significantly alter the response. Cultures treated with the two hydroxychalcones also displayed separate trends in ethoxyresorufin-O-deethylase (EROD) activity and DMBA–DNA covalent binding. In summary, the present study illustrated that the inhibition of hydroxychalcone on CYP1 enzymes and XRE-transactivation was affected by the position and number of hydroxyl groups in its structure.