Abstract: Objective To identify the existence of side population(SP) cells in human ovarian cancer cell line SKOV3 and to investigate whether SP cells have the biological characteristics of cancer stem cells. Methods SP and non-SP cells from SKOV3 were isolated by fluorescence-activated cell sorting after being stained by DNA-binding dye Hoechst 33342. Biological characteristics of SP and non-SP cells were evaluated, such as proliferation ability, colony-forming efficiency, invasion and migration ability, self-renewal ability and cell cycle. Results SP cells proportion in SKOV3 cell line was (1.12±0.104)%. The proliferation ability of SP cells was obviously greater than that of non-SP cells(P<0.05). The colony-forming efficiency of SP cells was also higher than that of non-SP cells(P<0.05). SP cells had a stronger ability of invasion and migration compared with non-SP cells(P<0.05). SP cells could differentiate into non-SP cells in vitro. Most of SP cells were in the G0/G1 phase. Conclusion SP cells in SKOV3 exhibit biological characteristics of cancer stem cells. The phenotype of SP cells could be used as a potentially useful tool for enriching ovarian cancer stem cells.