The prototype dioxin congener 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is known to exert anti-estrogenic effects via activation of the aryl hydrocarbon receptor (AhR) by interfering with the regulation of oestrogen homeostasis and the estrogen receptor α (ERα) signalling pathway. The AhR/ER cross-talk is considered to play a crucial role in TCDD- and E2-dependent mechanisms of carcinogenesis, though the concerted mechanism of action in the liver is not yet elucidated. The present study investigated TCDD's impact on the transcriptional cross-talk between AhR and ERα and its modulation by 17β-estradiol (E2) in the human hepatoma cell line HepG2, which is AhR-responsive but ERα-negative. Transient transfection assays with co-transfection of hERα and supplementation of receptor antagonists showed anti-estrogenic action of TCDD via down-regulation of E2-induced ERα signaling. In contrast, enhancement of AhR signaling dependent on ERα was observed providing evidence for increased cytochrome P450 (CYP) induction to promote E2 metabolism. However, relative mRNA levels of major E2-metabolizing CYP1A1 and 1B1 and the main E2-detoxifying catechol-O-methyltransferase were not affected by the co-treatments. This study provides new evidence of a TCDD-activated AhR-mediated molecular AhR/ERα cross-talk mechanism at transcriptional level via indirect inhibition of ERα and enhanced transcriptional activity of AhR in HepG2 cells.