Estrogen receptor alpha (ERa) is involved in numerous physiological and pathological processes, including breast cancer. Breast cancer therapy is therefore currently directed at inhibiting the transcriptional potency of ERa, either by blocking estrogen production through aromatase inhibitors or antiestrogens that compete for hormone binding. Due to resistance, new treatment modalities are needed and as ERa dimerization is essential for its activity, interference with receptor dimerization offers a new opportunity to exploit in drug design. Here we describe a unique mechanism of how ERa dimerization is negatively controlled by interaction with 14-3-3 proteins at the extreme C terminus of the receptor. Moreover, the small-molecule fusicoccin (FC) stabilizes this ERa/14-3-3 interaction. Cocrystallization of the trimeric ERa/14-3-3/FC complex provides the structural basis for this stabilization and shows the importance of phosphorylation of the penultimate Threonine (ERa-T594) for high-affinity interaction. We confirm that T594 is a distinct ERa phosphorylation site in the breast cancer cell line MCF-7 using a phospho-T594–specific antibody and by mass spectrometry. In line with its ERa/14-3-3 interaction stabilizing effect, fusicoccin reduces the estradiol-stimulated ERa dimerization, inhibits ERa/chromatin interactions and downstream gene expression, resulting in decreased cell proliferation. Herewith, a unique functional phosphosite and an alternative regulation mechanism of ERa are provided, together with a small molecule that selectively targets this ERa/14-3-3 interface.