Abstract The flavanone naringenin is known to possess only weak estrogenic properties, but some of its derivatives such as 8-prenylnaringenin are potent phytoestrogens. The aim of this study was to further clarify structure–function relationships of flavanones regarding their estrogenic or antiestrogenic properties by characterizing the new chemically synthesized naringenin derivative 7-( O-prenyl)naringenin-4′-acetate (7-O-PN). A yeast based reporter gene assay and MVLN cells, a MCF-7-derived cell line that possesses a luciferase reporter gene under the control of a vitellogenin estrogen responsive element, were used to investigate estrogenic actions of 7-O-PN in vitro. Estradiol (E2) has been used as a positive control. Subsequently a 3-day rat uterotrophic assay was performed to test for estrogenic effects. In addition, mRNA expression of estrogen sensitive genes in the uteri of these rats was measured using real time rtPCR. While E2 leads to a strong dose dependent signal in the yeast based reporter gene assay and in MVLN cells, 7-O-PN shows mild E2 antagonistic properties at concentrations 10 −8 and 10 −7 M, E2 agonistic properties at 10 −6 and 10 −5 M in MVLN cells and no effects on the yeast based system. In contrast to E2 treatment, 7-O-PN treatment did not increase uterus wet weight compared to the negative control. These findings are supported by mRNA expression studies of proliferation markers. Additionally, mRNA expression studies of estrogen regulated genes revealed very strong antiestrogenic properties of 7-O-PN regarding regulation of complement C3 expression while some estrogenic effects could be observed on the expression of estrogen receptor β, clusterin and possibly on progesterone receptor and vascular endothelial growth factor.