MicroRNAs (miRNAs) are a class of small noncoding RNAs that control gene expression by targeting mRNAs and triggering either translational repression or RNA degradation. The aberrant expression of miRNAs might be involved in human diseases, including cancer. The expression of miR-206 in estrogen receptor alpha (ER-α)-positive human breast cancer tissues is well known. However, the expression and regulation of miR-206 in the developing mammary gland has not yet been studied. To understand the effects of miR-206 on mammary gland development, we have profiled gene expression in scramble-transfected and miR-206-overexpressing developing mammary buds. The genes that are potentially regulated by miR-206 in the mammary epithelium and/or mesenchyme, such as Tachykinin1 and Gata3, are known to be breast cancer markers. The expression of Wnt, which is involved in gland positioning, and of the transcription factors Tbx3 and Lef1, which are essential for mammary gland development, changes after miR-206 overexpression. Using a mammary bud in vitro culture system, we have demonstrated that miR-206 acts downstream of ER-α during mammary gland growth. Thus, miR-206 might be a novel candidate for morphogenesis during the initiation of mammary gland formation and the regulation of genes related to mammary gland development and breast cancer.