The state of cancer stem cells (CSC) under reversible fluctuations, which has been revealed in breast cancer cells most recently, suggests that subpopulations with distinct phenotypes and functions within cancer cells can undergo inter-conversion. To investigate the possibility in colon cancer cells, we employed CD133 as the CSC marker, and characterized CD133 expression pattern and the biological features of the CD133 (+) and CD133 (-) subsets. Flow cytometry revealed that CD133 was bimodally expressed in SW620 cells among eight colon cancer cell lines. The CD133 (+) clonal SW620 cells displayed a differential gene expression profile, higher cellular reactive oxygen species (ROS), enhanced tumorigenesis and resistance to 5-fluorouracil. The conversion in term of the CD133 phenotype of the sorted cells was observed in vitro and in vivo. The fraction of the CD133 (+) cells decreased from 99% to 80% in the sorted CD133 (+) population while rising from 5 to 10% in the sorted CD133 (-) population during the first 20-day cultivation and then stayed almost unchanged. A fraction (about 20%) of the CD133 (+) clonal cells lost their CD133 marker while about 10% of the CD133 (-) clonal cells acquired the CD133 marker. 5-Azacytidine enhanced the fraction of the CD133 (+) cells in both of the CD133 (+) and CD133 (-) clonal cells. Our data demonstrate that CD133 expression is dynamic and reversible, and reveal the inter-conversion between the CD133 (+) and the CD133 (-) SW620 cells, suggesting that the CD133 phenotype of SW620 cell population is retained by the conversion between the two cell subsets.