A ligand-dependent nuclear transcription factor, ERα has two transactivating functional domains (AF), AF-1 and AF-2. AF-1 is localized in the N-terminal region and AF-2 is distributed in the C-terminal ligand-binding domain (LBD) of the ERα protein. Helix 12 (H12) in the LBD is a component of the AF-2 and the configuration of H12 is ligand inducible to an active or inactive form. Previously, we demonstrated that the ERα mutant (AF2ER) possessing L543A, L544A mutations in H12 disrupts AF-2 function and reverses antagonists such as fulvestrant/ICI182780 (ICI) or 4-hydoxytamoxifen (OHT) into agonists, in the AF2ER knock-in mouse. Our previous in vitro studies suggested that the mode of AF2ER activation is similar to the partial agonist activity of OHT for WT-ERα. However it is still unclear how antagonists activate ERα. To understand the molecular mechanism of antagonist reversal activity, we analyzed the correlation between the ICI dependent ERE-mediated transcription activity of AF2ER and AF2ER-LBD dimerization activity. We report here that ICI dependent AF2ER activation correlated with the activity of AF2ER-LBD homodimerization. Prevention of dimerization impaired the ICI dependent ERE binding and transcription activity of AF2ER. The dislocation of H12 caused ICI dependent LBD homodimerization involving the F-domain, the adjoining region of H12. Furthermore, F-domain truncation also strongly depressed the dimerization of WT-ERα-LBD with antagonists but not with E2. AF2ER activation levels with ICI, OHT and raloxifene were parallel with the degree of AF2ER-LBD homodimerization; supporting a mechanism that antagonist-dependent LBD homodimerization involving the F-domain results in antagonist reversal activity of ERα.