Background: Diarylheptanoid (D3) isolated from the medicinal plant, Curcuma comosa, has estrogenic activity.Objective: We aimed to elucidate the mechanism(s) of D3 action and compare it with that of 17β-estradiol (E2) using both in vitro and in vivo uterine models.Methods: We used human uterine (Ishikawa) cells to determine the estrogenic action of D3 on the activation and nuclear translocation of estrogen receptor α (ERα). In addition, we further characterized the uterine response to D3 treatment in vivo.Results: D3 activated an estrogen responsive element (ERE) luciferase reporter through ERα, and molecular modeling suggested that D3 could be accommodated in the ERα binding pocket. Using modified ERα to assay ligand-dependent nuclear translocation, we observed D3-dependent ERα interaction and translocation. In mouse uteri, early- and late-phase estrogen-regulated gene responses were increased in D3-treated ovariectomized wild-type animals, in a manner similar to that of E2; no response was seen in ERα knockout animals. We observed a divergence in estrogen responses after D3 treatment: D3 induced robust DNA synthesis in uterine epithelial cells, linked to an increase in cell-cycle–related genes; however, no increase in uterine weight was observed 24 hr after treatment. D3 also affected uterine progesterone receptor expression patterns similar to E2. When D3 and E2 were administered together, we observed no additive or antagonistic effects of D3 on E2. Our findings suggest that D3 is a weak estrogenic agonist compound.Conclusion: D3 is a weakly acting phytoestrogen that mimics the mitogenic responses produced by E2 in an ERα-dependent manner, but it is unable to increase uterine weight or enhance or antagonize the effects of estrogen.