Breast cancers are sensitive to hormones such as estrogen, which binds to and activates estrogen receptors (ER) leading to significant changes in gene expression. microRNAs (miRNA) have emerged as a major player in gene regulation, thus identification of miRNAs associated with normal or disrupted estrogen signaling is critical to enhancing our understanding of the diagnosis and prognosis of breast cancer. We have previously shown that 17β-estradiol (E2) induced activation of ERα in T47D cells results in significant changes in the expression of protein-coding genes involved in cell cycle, proliferation, and apoptosis. To identify miRNAs regulated by E2-activated ERα, we analysed their expression in T47D cells following E2-activation using both dual-color microarrays and TaqMan Low Density Arrays, and validations were carried out by real-time PCR. Although estrogen treatment results in altered expression of up to 900 protein-coding transcripts, no significant changes in mature miRNA expression levels could be confirmed. Whereas previous studies aiming to elucidate the role of miRNA in ER-positive breast cancers cell lines have yielded conflicting results, the work presented here represents a thorough investigation of and significant step forward in our understanding of ERα mediated miRNA regulation.