Tamoxifen is one of the major drugs used for the hormonotherapy of estrogen receptor positive breast cancers. However, its therapeutic efficacy can be limited by acquired resistance and tumor recurrence can occur after several years of treatment. Tamoxifen is known as the prototypical modulator of estrogen receptors, but other targets have been identified that could account for its pharmacology. In particular, tamoxifen binds with high affinity to the microsomal antiestrogen binding site (AEBS) and inhibits cholesterol esterification at therapeutic doses. We have recently shown that the AEBS was a hetero-oligomeric complex composed of 3β-hydroxysterol-Δ(8)-Δ(7)-isomerase and 3β-hydroxysterol-Δ(7)-reductase, that binds different structural classes of ligands, including selective estrogen receptor modulators, several sigma receptor ligands, poly-unsaturated fatty acids and ring B oxysterols. We established a link between the modulation of cholesterol metabolism by tamoxifen and other AEBS ligands and their capacity to induce breast cancer cell differentiation, apoptosis and autophagy. Moreover, we showed that the AEBS carries out cholesterol-5,6-epoxide hydrolase activity and established that cholesterol-5,6-epoxide hydrolase is a new target for tamoxifen and other AEBS ligands. Finally in this review, we report on recent data from the literature showing how the modulation of cholesterol and oxysterol metabolism can be linked to the antitumor and chemopreventive properties of tamoxifen, and give new perspectives to improve the clinical outcome of the hormonotherapy of breast cancers.