Numerous dietary compounds can modify gene expression by binding to the members of the nuclear receptor superfamily of transcription factors. For example, dietary polyphenols, such as soy isoflavones genistein and daidzein, modulate the activity of the estrogen receptors ER and ER. An additional class of dietary polyphenols that modulate cellular signaling pathways are lignans, compounds that are common constituents of Western diets. In this study, we show that a metabolite of dietary lignans, enterolactone, at physiological concentrations, activates ER-mediated transcription in vitro with preference for ER. The effects of enterolactone are mediated by the ER ligand binding domain and are susceptible to antiestrogen treatment. Furthermore, the affinity of enterolactone towards ER, measured by a novel ligand binding assay, is augmented in cell culture conditions. Moreover, our results demonstrate for the first time that enterolactone has estrogenic activity in vivo. In transgenic estrogen sensitive reporter mice, enterolactone induces tissue-specific estrogen responsive reporter gene expression, as well as promotes uterine stromal edema and expression of estrogen responsive endogenous genes (CyclinD1 and Ki67). Taken together, our data shows that enterolactone is a selective ER agonist inducing ER-mediated transcription both in vitro in different cell lines and in vivo in the mouse uterus.