In this study we investigated the impact of estrogen antagonists and of 16α-OHE1 (an estrogen derivative that binds to and induces transactivation of estrogen receptors) on estrogen metabolism in malignant HepG2 human liver cells featured by high estrogen sulfotransferase (EST); our aim was to clarify the potential correlation of EST and ER. As expected, the HepG2 cells exhibited a very high EST activity, with the majority of estrogen metabolites (over 86%) being detected as sulfates by 24 h. The coincubation of E2 and the antiestrogen tamoxifen induced a weak inhibition of EST activity (from 85.4% to 81.5%), while the coincubation with the pure antagonist ICI-182 and with 16α-OHE1 produced a 50% and 90% decrease of EST, respectively. Interestingly, both selective estrogen receptor modulators (SERMs) TAM and ICI-182, along with the same 16α-OHE1, gave rise respectively to a 2.8%, 3.2%, and 4.6% of de novo 16α-OHE1 formation. The inhibition of EST and the increase of 16α-OHE1 formation were both time- and dose-dependent. Our results suggest that EST activity is tightly associated with ER transactivation and can be regulated by selective estrogen receptor modulators (SERMs), including antiestrogens and 16α-OHE1. In this framework, 16α-OHE1 may have a potential role in human liver carcinogenesis, also through the inhibition of EST and the production of unconjugated, bioavailable estrogens.