To characterize the biological features of side population (SP) in oral squamous cell carcinoma (OCC), SP and non-SP were sorted and compared. The SP cells were more clonogenic and in nude mice, only 10,000 SP cells were needed for tumor development compared to 1,000,000 non-SP cells. The SP cells expressed higher levels of ABCG2, ABCB1, CD44, Oct-4, Bmi-1, NSPc1 and CK19. The SP cells generated SP and non-SP populations, whereas the non-SP cells generated only non-SP. These findings provide the first evidence that SP in OCC possesses tumor stem cell phenotypes and may play an important role in OCC tumorigenesis.