Estetrol (E 4) is a natural estrogen with a long half-life produced only by the human fetal liver during pregnancy. The crystal structures of the estrogen receptor α (ERα) ligand-binding domain bound to 17β-estradiol (E 2) and E 4 are very similar, as well as their capacity to activate the two activation functions AF-1 and AF-2 and to recruit the coactivator SRC3. In vivo administration of high doses of E 4 stimulated uterine gene expression, epithelial proliferation, and prevented atheroma, three recognized nuclear ERα actions. However, E 4 failed to promote endothelial NO synthase activation and acceleration of endothelial healing, two processes clearly dependent on membrane-initiated steroid signaling (MISS). Furthermore, E 4 antagonized E 2 MISS-dependent effects in endothelium but also in MCF-7 breast cancer cell line. This profile of ERα activation by E 4, uncoupling nuclear and membrane activation, characterizes E 4 as a selective ER modulator which could have medical applications that should now be considered further.