Synthetic progesterone used in contraception drugs (progestins) can promote breast cancer growth but the mechanisms involved are unknown. Moreover, it remains unclear whether cytoplasmic interactions between the progesterone receptor (PR) and estrogen receptor alpha (ERα) is required for PR activation. In this study, we used a murine progestin-dependent tumor to investigate the role of ERα in progestin-induced tumor cell proliferation. We found that treatment with the progestin medroxyprogesterone acetate (MPA) induced the expression and activation of ERα, as well as rapid nuclear co-localization of activated ERα with PR. Treatment with the pure anti-estrogen fulvestrant to block ERα disrupted the interaction of ERα and PR in vitro and induced the regression of MPA-dependent tumor growth in vivo. ERα blockade also prevented an MPA-induced increase in CYCLIN D1 (CCND1) and MYC expression. Chromatin immunoprecipitation studies demonstrated that MPA triggered binding of ERα and PR to the CCND1 and MYC promoters. Interestingly, blockade or RNAi-mediated silencing of ERα inhibited ERα, but not PR binding to both regulatory sequences, indicating that an interaction between ERα and PR at these sites is necessary for MPA-induced gene expression and cell proliferation. We confirmed that nuclear co-localization of both receptors also occurred in human breast cancer samples. Together, our findings argued that ERα-PR association on target gene promoters is essential for progestin-induced cell proliferation.