Botanical dietary supplements (BDS) containing hops are sold as women's health supplements due to the potent hop phytoestrogen, 8-prenylnaringenin (8-PN), and the cytoprotective chalcone, xanthohumol. Previous studies have shown a standardized hop extract to beneficially influence chemical estrogen carcinogenesis in vitro by fostering detoxified 2-hydroxylation over genotoxic 4-hydroxylation estrogen metabolism. In this study, hop extract and its bioactive compounds were investigated for its mechanism of action within the chemical estrogen carcinogenesis pathway, which is mainly mediated through the 4-hydroxylation pathway catalyzed by CYP1B1 that can form gentoxic quinones. Aryl hydrocarbon receptor (AhR) agonists induce CYP1A1 and CYP1B1, while estrogen receptor alpha (ERα) inhibits transcription of CYP1A1, the enzyme responsible for 2-hydroxylated estrogens and the estrogen detoxification pathway. An In-Cell Western MCF-7 cell assay revealed hop extract and 6-prenylnaringenin (6-PN) degraded ERα via an AhR-dependent mechanism. Reverse transcription PCR and xenobiotic response element luciferase assays showed hop extract and 6-PN-mediated activation of AhR and induction of CYP1A1. A reduction in estrogen-mediated DNA (cytosine-5)-methyltransferase 1 (DNMT1) downregulation of CYP1A1 accompanied this activity in a chromatin immunoprecipitation assay. Ultimately, hop extract and 6-PN induced preferential metabolism of estrogens to their detoxified form in vitro. These results suggest that the standardized hop extract and 6-PN activate AhR to attenuate epigenetic inhibition of CYP1A1 through degradation of ERα, ultimately increasing 2-hydroxylated estrogens. A new mechanism of action rationalizes the positive influence of hop BDS and 6-PN on oxidative estrogen metabolism in vitro and, thus, potentially on chemical estrogen carcinogenesis. The findings underscore the importance of elucidating various biological mechanisms of action and standardizing BDS to multiple phytoconstituents for optimal resilience promoting properties.