Abstract: Objective To sort side population(SP)cells in human colorectal cancer tissues and explore its related biological characteristics. Methods SP cells were selected by Hoechst33342 staining and fluorescence activated cell sorting(FACS)technoligy, and non-side population(SP)cells were used as control. Three dimensional(TD)cultures of SP cells were used to analyze their tumorigenic ability in nude mice. The spheroidizing abilities of SP cells and NSP cells were observed in serum-free culture, and the differences of drug resistance between SP cells and NSP cells were analyzed by water soluble tetrazolium salt reagent(WST). Results The proportion of SP cells in human colorectal cancer was(0.90±0.10)%. SP cells in serum-free medium had better ability to form suspension cells than NSP cells. When 10³, 10⁴, 10⁵ and 10⁶ SP cells were inoculated subcutaneously in nude mice, the tumorigenesis rates were 30.0%, 50.0%, 70.0% and 100.0% respectively, while the tumorigenesis rates of NSP cells were 0%, 0%, 40.0% and 60.0% respectively, and there was significant difference in tumorigenesis rate between SP cells and NSP cells(P<0.05). The resistance rate of SP cells to 5-FU was significantly higher than that of NSP cells(P<0.05). Conclusions There are SP cells in human colorectal cancer tissue. SP cells have stem cell characteristics. SP cells can exist as cell spheres in serum-free medium. SP cells are resistant to 5-FU.