Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer-related mortality in females worldwide. The efficacy of chemotherapeutic drugs on breast cancer is hindered by many factors, including that cancer stem-like side population (SP) cells may contribute to tumorigenesis and resistance to chemotherapy. Doxorubicin and gemcitabine are two of the most effective and widely used chemotherapeutic agents for the treatment of various human malignancies. To the best of our knowledge, the effects of doxorubicin and gemcitabine on breast cancer SP cells are poorly understood. In the present study, we examined the potential roles of doxorubicin and gemcitabine in breast cancer main population (MP) and SP cells, and the mechanisms of doxorubicin and gemcitabine. The results showed that doxorubicin and gemcitabine decreasede proliferation, and increased apoptosis in the MCF7 MP and SP cells in vitro. Furthermore, doxorubicin and gemcitabine inhibited tumor growth and improved the survival rate of mice in vivo. Additionally, the mechanisms of doxorubicin and gemcitabine in MCF7 MP and SP cells were determined.