microRNAs (miRNAs) are involved in controlling tumor behaviors either as oncogenes or tumor suppressors. To elucidate the role of miRNAs in the regulation of tumor initiation, we delineated the microRNA expression signature characterizing the properties of tumor-initiating cells for breast cancer. A group of miRNAs were differentially expressed in MDA-MB-231 and SUM1315 cells (with a high proportion of breast cancer tumor-initiating cells, CD44(+)CD24(-/low) subpopulation) compared to MCF-7 cells (only a small proportion of CD44(+)CD24(-/low) cells). Among the differentially expressed miRNAs common to MDA-MB-231 and SUM1315, approximately 46% of them are suggested to regulate the 'stemness' of stem cells or progenitor cells. Taken together, these findings suggested that miRNAs contribute to the maintenance of tumor-initiating properties and indicate the potential value of the miRNA expression signature in characterizing or predicting the features (including metastasis) of breast cancer.