We previously showed that COUP-TFI interacts with the Estrogen Receptor alpha (ER alpha) to recruit Extracellular signal Regulated Kinases (ERKs) in an Estradiol (E2)-independent manner, resulting in an enhancement of ER alpha transcriptional activity. However, the involvement of COUP-TFI in physiologically relevant functions of ER alpha, such as the mitogenic activity that E2 has on breast cancer cells, remains poorly understood. Here, we first showed that the amounts of COUP-TFI protein are higher in dedifferentiated mammary cell lines (MDA-MB-231) and tumor breast cells as compared to the differentiated MCF-7 cell line and normal breast cells. To evaluate the functional relevance of the COUP-TFI/ER alpha interplay in mammary cells, we generated MCF-7 cells that stably over-express COUP-TFI. We found that the over-expression of COUP-TFI enhances motility and invasiveness of MCF-7 cells. COUP-TFI also promotes the proliferation of MCF-7 cells through ER alpha-dependent mechanisms that target cell cycle progression and cell survival. To further investigate the mechanisms underlying these effects of COUP-TFI, we evaluated the expression of known E2-target genes in breast cancer, and found that COUP-TFI differentially regulated genes involved in cell proliferation, apoptosis, and migration/invasion. Notably, Cathepsin D (CTSD) transcript and protein levels were significantly higher in presence and absence of E2 in MCF-7 over-expressing COUP-TFI. Chromatin Immunoprecipitation assays showed that ER alpha, phospho-RNA Polymerase II, as well as p68 RNA Helicase, a phospho-Serine 118 dependent co-activator of ER alpha, were preferentially recruited onto the CTSD gene proximal promoter in COUP-TFI over-expressing cells. These results suggest that COUP-TFI selectively regulates the expression of endogenous E2-target genes and consequently modifies ER alpha positive mammary cells response to E2.