The major goal of breast cancer prevention is to reduce the incidence of ductal carcinoma in situ (DCIS), an early stage of breast cancer. However, the biology behind DCIS formation is not well understood. It is suspected that cancer stem cells (CSCs) are already programmed in pre-malignant DCIS lesions and that these tumor-initiating cells may determine the phenotype of DCIS. MicroRNA (miRNA) profiling of paired DCIS tumors revealed that loss of miR-140 is a hallmark of DCIS lesions. Previously, we have found that miR-140 regulates CSCs in luminal subtype invasive ductal carcinoma. Here, we find that miR-140 has a critical role in regulating stem cell signaling in normal breast epithelium and in DCIS. miRNA profiling of normal mammary stem cells and cancer stem-like cells from DCIS tumors revealed that miR-140 is significantly downregulated in cancer stem-like cells compared with normal stem cells, linking miR-140 and dysregulated stem cell circuitry. Furthermore, we found that SOX9 and ALDH1, the most significantly activated stem-cell factors in DCIS stem-like cells, are direct targets of miR-140. Currently, targeted therapies (tamoxifen) are only able to reduce DCIS risk in patients with estrogen receptor α (ERα)-positive disease. We examined a model of ERα-negative/basal-like DCIS and found that restoration of miR-140 via a genetic approach or with the dietary compound sulforaphane decreased SOX9 and ALDH1, and reduced tumor growth in vivo. These results support that a miR-140/ALDH1/SOX9 axis is critical to basal CSC self-renewal and tumor formation in vivo, suggesting that the miR-140 pathway may be a promising target for preventative strategies in patients with basal-like DCIS.