목적: Radioresistance is one of the main determinants of treatment outcome in head and neck squamous cell carcinoma (HNSCC). We aimed to isolate cancer stem cells (CSC) from radioresistant HNSCC to investigate the role of Nanog in the maintenance of CSC and causative effect of CSC in the radioresistance. 방법:To induce radioresistant cell lines, we treated SCC15, SCC25 and QLL1 with an accumulated dosage of 60 Gy over 30 cycles of irradiation. Radioresistant cells were cultured in the serum free media on ultra-low attachment culture flask to induce sphere- forming cells. CSC characteristics of sphere-forming cells were verified by western blot for CD-44, Oct-4, Nanog, Sox-2 and CD-133. After inhibition of Nanog in CSC, we observed the change of characteristics and radioresistance of CSC. 결과:Sphere-forming cells were identified in all three cancer cell lines and it took 3-5 days after serum depravation. All sphere-forming cells from three different cancer cell lines expressed stem cell marker, however they showed some different extent of expressions. CSCs showed more radioresistance than monolayer cells (culture with serum) after irradiation by MTT assay and colony forming assay. Knock out of Nanog in the CSCs showed reduced radioresistance as well as decreased size of sphere, low expression of CSC markers, less invasion and migration of radioresistant CSCs comparing to Nanog expressing CSC. 결론:CSCs seem to be the cause of radioresistant property in the radioresistant HNSCC. Nanog may have a key role in maintaining radioresistance as well as CSC property and it may be used in target therapy in the radioresistant head and neck cancer.