Objective At present, the methods of separating and identifying nasopharyngeal cancer stem cells are not yet mature.This study was to explore the methods of culturing nasopharyngeal cancer stem cell microspheres and identify the cancerous stem cell biological features of CNE-2 cell microspheres. Methods We conducted suspension culture of human nasopharyngeal cancer CNE2 cells and C666-1 cells in serum-free medium ( SFM) containing growth factors.Then we measured the proportion of CD133 +cells in CNE2 monolayer ( CNE2-MN) and CNE2 microsphere cells ( CNE2-SC) by flow cytometry, determined their in vitro invasiveness through Transwell chamber experiments, and detected their in vivo tumorigenicity via nude mouse experiments.We ob-served the differentiation potency of the CNE2-SCs in the adherent-cultured serum-containing medium and detected the expressions of the cancer stem cell-related genes Bmi-1, Oct4, and Twist1 in CNE2-MN and CNE2-SCs by flow cytometry and RT-PCR analysis. Results In the special blend of SFM, both of the cell lines can form microspheres that can be stably transferred.Fresh SFM prepara-tion, substituting cell separation agent Accutase for pancreatic enzyme for transfer, and maintaining the state of cell suspension contrib-uted to the formation and proliferation of microspheres.Adherent culture with serum-containing medium induced the differentiation of CNE2-MN cells, which exhibited no significant difference from the CNE2 microsphere cells.The CD133 +cells accounted for 98.79%in the CNE2 microspheres, significantly higher than 0.98%in the CNE2 cells (P<0.01).Compared with the CNE2-MN cells, the CNE2-SCs showed highly increased expressions of Bmi-1, Oct4, and Twist1 (P<0.01).The numbers of membrane-penetrating cells in the CNE2-SCs and CNE2-MN cells were 122 ±6 and 36 ±7 per visual field, the former with a stronger invasive ability than the latter genesis of 1 ×106 CNE2-SCs vs that of the same number of CNE2 -MNcells was (2.332 ±0.549) cm 3 sv (0.669 ±0 .278) cm3 ( P<0.01). Conclusion Using suspension culture with a specially prepared SFM, nasopharyngeal cancer CNE2 microsphere cells can be obtained, in which there are large numbers of cancer stem cells.This culture method may provide a base for further studies of naso-pharyngeal cancer stem cells.