Abstract: Objective To isolate colon cancer stem cells from human colorectal carcinoma and study the biological characteristics in vitro. Methods Primary colorectal carcinoma cells were cultured in serum free conditions which give rise preferentially to self-renewal stem cell spheres. Cells have capability of developing into tumor spheres were cloned by limiting dilution and clones from single-cell proliferation were isolated. The proliferative potential of cloned cells was tested using MTT colorimetric method. Expression of cell surface marks, including CD133, CD166, CD24, CD47, CD200, CD90, CD44 and EPCAM were analyzed by fl ow cytometry. The expression of stem cell-specifi c genes, Oct4, Sox2, C-myc and Nanog, by cell clones was tested and quantifi ed by real-time quantitative PCR. Results From primary cultures, 10 clones which were capable of unlimited self-renewal were isolated and expended by continuous subcultures. These clones were positive for surface marks CD166, CD47, CD44, and CD90, and negative for CD133, CD24, CD200 and EPCAM. Cell proliferation analysis showed that, comparing with their original cultures, cloned cells had higher proliferation rate and expressed higher levels of stem cell-specifi c genes Oct4, Sox2 and C-myc. Expression of Nanog was not detected in both cloned cells and their original cultures. Conclusion Colon cancer stem cells existing in human colorectal carcinoma have biological characteristics of self-renewing and indefi nite proliferation. Those tumor cells could be isolated, culture and purifi ed in vitro.