A major obstacle in the successful treatment of cancer is the occurrence of chemoresistance. Cancer cells surviving chemotherapy and giving rise to a recurrence of the tumor are termed cancer stem cells and can be identified by elevated levels of certain stem cell markers. Eradication of this cell population is a priority objective in cancer therapy. Here, we report elevated levels of stem cell markers in MCF-7 mammospheres. Likewise, an upregulation of HER2 and its differential expression within individual cells of mammospheres was observed. Sorting for HER2high and HER2low cells revealed an upregulation of stem cell markers NANOG, OCT4 and SOX2 in the HER2low cell fraction. Accordingly, HER2low cells also showed reduced proliferation, ductal-like outgrowths and an increased number of colonies in matrigel. Xenografts from subcutaneously injected HER2low sorted cells exihibited earlier onset but slower growth of tumors and an increase in stem cell markers compared to tumors developed from the HER2high fraction. Treatment of mammospheres with salinomycin reduced the expression of SOX2 indicating a selective targeting of cancer stem cells. Trastuzumab however, did not reduce the expression of SOX2 in mammospheres. Furthermore, a combinatorial treatment of mammospheres with trastuzumab and salinomycin was superior to single treatment with each drug. Thus, targeting HER2 expressing tumors with anti-HER2 therapies will not necessarily eliminate cancer stem cells and may lead to a more aggressive cancer cell phenotype. Our study demonstrates efficient killing of both HER2 positive cells and cancer stem cells, hence opening a possibility for a new combinatorial treatment strategy.