The primary induced isoflavones in soybean, the glyceollins, have been shown to be potent estrogen antagonists in vitro and in vivo. The discovery of the glyceollins’ ability to inhibit cancer cell proliferation has led to the analysis of estrogenic activities of other induced isoflavones. In this study, we investigated a novel isoflavone, glycinol, a precursor to glyceollin that is produced in elicited soy. Sensitive and specific in vitro bioassays were used to determine that glycinol exhibits potent estrogenic activity. Estrogen-based reporter assays were performed, and glycinol displayed a marked estrogenic effect on estrogen receptor (ER) signaling between 1 and 10 µM, which correlated with comparable colony formation of MCF-7 cells at 10 µM. Glycinol also induced the expression of estrogen-responsive genes (progesterone receptor and stromal-cell-derived factor-1). Competitive binding assays revealed a high affinity of glycinol for both ER α (IC50 = 13.8 nM) and ER β (IC50 = 9.1 nM). In addition, ligand receptor modeling (docking) studies were performed and glycinol was shown to bind similarly to both ER α and ER β. Taken together, these results suggest for the first time that glycinol is estrogenic and may represent an important component of the health effects of soy-based foods.