Trastuzumab-refractory breast cancer stem cells (CSCs) could explain the high rate of primary resistance to single-agent trastuzumab in HER2 gene-amplified breast cancer patients. The identification of agents with strong selective toxicity for trastuzumabresistant breast CSCs may have tremendous relevance for how HER2+ breast cancer patients should be treated. Using the human breast cancer cell line JIMT-1, which was established from the pleural metastasis of a patient who was clinically resistant to trastuzumab ab initio, we examined whether preferential killing of the putative CD44+CD24-/low breast CSC population might be sufficient to overcome primary resistance to trastuzumab in vivo. Because recent studies have shown that the antidiabetic biguanide metformin can exert antitumor effects by targeted killing of CSClike cells, we explored whether metformin’s ability to preferentially kill breast cancer initiating CD44+CD24-/low cells may have the potential to sensitize JIMT-1 xenograft mouse models to trastuzumab. Upon isolation for breast cancer initiating CD44+CD24-/ low cells by employing magnetic activated cell sorting, we observed the kinetics of metformin-induced killing drastically varied among CSC and non-CSC subpopulations. Metformin’s cell killing effect increased dramatically by more than 10-fold in CD44+CD24-/low breast CSC cells compared to non-CD44+CD24-/low immunophenotypes. While seven-weeks treatment length with trastuzumab likewise failed to reduce tumor growth of JIMT-1 xenografts, systemic treatment with metformin as single agent resulted in a significant two-fold reduction in tumor volume. When trastuzumab was combined with concurrent metformin, tumor volume decreased sharply by more than four-fold. Given that metformin-induced preferential killing of breast cancer initiating CD44+CD24-/low subpopulations is sufficient to overcome in vivo primary resistance to trastuzumab, the incorporation of metformin into trastuzumab-based regimens may provide a valuable strategy for treatment of HER2+ breast cancer patients