Breast cancer is the most frequently diagnosed malignancy and the leading cause of cancer-associated death in women worldwide. microRNAs (miRNAs) play critical roles in the cellular processes of breast cancer. However, the crucial roles and underlying mechanisms of miR-539 in breast cancer remain unclear. By RT-qPCR, we found that expression of miR-539 was markedly down-regulated in breast cancer tissues and cell lines compared with that in paired adjacent normal tissues and normal cell lines. The low level of miR-539 expression was positively associated with lymph node metastasis. Furthermore, forced expression of miR-539 inhibited proliferation and migration of breast cancer MDA-MB-231 and MCF7 cells in vitro and suppressed tumor growth in vivo. Moreover, bioinformatics analysis and luciferase reporter assays indicated that epidermal growth factor receptor (EGFR) was a direct target of miR-539. Over-expression of miR-539 decreased the EGFR mRNA and protein levels in MDA-MB-231 and MCF7 cells. In addition, ectopic over-expression of EGFR partly reversed miR-539-inhibited proliferation as well as migration of MDA-MB-231 and MCF7 cells. Taken together, our results demonstrate that miR-539 functions as a tumor suppressor in breast cancer by downregulating EGFR, supporting the targeting of the novel miR-539/EGFR axis as a potentially effective therapeutic approach for breast cancer.