Breast cancer is the most common type of malignancy among females. Previous studies examining breast cancer tissue have demonstrated the presence of stem cells, and have detected octamer‑binding protein 4 (Oct4) and Nanog transcription factor expression. In the present study, breast cancer stem cells (CSCs) were isolated and enriched from MDA‑MB‑231 breast cancer cell lines, and were defined as MDA‑MB‑231 stem cells using flow cytometry. The expression of Oct4 and Nanog in breast CSCs were detected by quantitative polymerase chain reaction and western blotting. RNA interference (RNAi) was used in order to downregulate the expression of Oct4 and Nanog. Drug resistance and tumor‑initiating capability following in vivo injection of MDA‑MB‑231 stem cells trans-duced with negative RNAi, Oct4 RNAi and Nanog RNAi were compared with that of MDA‑MB‑231 stem cells without siRNA transfection as a control group. In addition the capability of MDA‑MB‑231 breast cancer cells to initiate tumor formation in mice was compared with that of MDA‑MB‑231 stem cells. A paclitaxel inhibition test was also conducted in order to detect resistance of MDA‑MB‑231 breast cancer stem cells to this treatment. The MDA‑MB‑231 stem cells were revealed to exhibit elevated percentages of the cluster of differentiation (CD)44+CD24‑/low subset, high tumorigenicity and resistance to chemotherapy, all of which are characteristic stem cell properties. In addition, the MDA‑MB‑231 stem cells were more tumorigenic in vivo. Furthermore, the breast CSCs also expressed high levels of the Oct4 and Nanog transcription factors. Therefore, downregulation of Oct4 or Nanog expression may reduce chemotherapeutic drug resistance and tumorigenicity in breast CSCs. In conclusion, Oct4 and Nanog expression may be a key factor in the development of resistance to chemotherapy and tumor growth of breast CSCs. This finding indicates that Oct4 or Nanog‑targeted therapy may be a promising means of overcoming resistance to chemotherapy and inhibiting tumor growth in breast cancer treatment.