CD133 (prominin-1), a 5-transmembrane glycoprotein, has recently been considered to be an important marker that represents the subset population of cancer stem-like cells. Herein we report the isolation of CD133-positive cells (LC-CD133⁺) and CD133-negative cells (LC-CD133⁻) from tissue samples of ten patients with non-small cell lung cancer (LC) and five LC cell lines. LC-CD133⁺ displayed higher Oct-4 expressions with the ability to self-renew and may represent a reservoir with proliferative potential for generating lung cancer cells. Furthermore, LC-CD133⁺, unlike LC-CD133⁻, highly co-expressed the multiple drug-resistant marker ABCG2 and showed significant resistance to chemotherapy agents (i.e., cisplatin, etoposide, doxorubicin, and paclitaxel) and radiotherapy. The treatment of Oct-4 siRNA with lentiviral vector can specifically block the capability of LC-CD133⁺ to form spheres and can further facilitate LC-CD133⁺ to differentiate into LC-CD133⁻. In addition, knock-down of Oct-4 expression in LC-CD133⁺ can significantly inhibit the abilities of tumor invasion and colony formation, and increase apoptotic activities of caspase 3 and poly (ADP-ribose) polymerase (PARP). Finally, in vitro and in vivo studies further confirm that the treatment effect of chemoradiotherapy for LC-CD133⁺ can be improved by the treatment of Oct-4 siRNA. In conclusion, we demonstrated that Oct-4 expression plays a crucial role in maintaining the self-renewing, cancer stem-like, and chemoradioresistant properties of LC-CD133⁺. Future research is warranted regarding the up-regulated expression of Oct-4 in LC-CD133⁺ and malignant lung cancer.