Hops (Humulus lupulus) is used as an alternative to hormone replacement therapy due to the phytoestrogen, 8-prenylnaringenin (8-PN). To examine the potential risks/benefits of hops extract and its compounds (8-PN and 6-prenylnaringenin, 6-PN), we aimed to evaluate the estrogen receptor α (ERα) and aryl hydrocarbon receptor (AHR) signaling pathways in human endometrial cancer cells. Hops extract, 8-PN and 6-PN showed estrogenic activity. Hops extract and 6-PN activated both ERα and AHR pathways. 6-PN increased the expression of the tumor suppressor gene (AHRR), and that of genes involved in the estrogen metabolism (CYP1A1, CYP1B1). Although 6-PN might activate the detoxification and genotoxic pathways of estrogen metabolism, hops extract as a whole only modulated the genotoxic pathway by an up-regulation of CYP1B1 mRNA expression. These data demonstrate the relevant role of 6-PN contained in the hops extract as potential modulator of estrogen metabolism due to its ERα and AHR agonist activity.