Worldwide, colorectal cancer is the third most common type of cancer affecting both sexes. It has been proposed that a small subset of cancer cells (cancer stem cells) within each tumor is able to initiate tumor growth. In 2007, two research groups simultaneously identified a colon cancer stem cell population in human tumors by the use of CD133 expression. In the present study, we used a human colon cancer cell line, SW620, to analyze the cancer stem cell-like characteristics of CD133(+) cells in vitro and in vivo. In vitro, CD133(+) SW620 cells had a higher proliferative capacity, were more irradiation- and chemotherapy-resistant, and had a higher expression of β-catenin compared with CD133(-) cells. Injections of either CD133(+) or CD133(-) cells into the skin or rectal mucosa of NOD/SCID mice led to tumors; however, injection of CD133(+) cells resulted in the formation of larger tumors. Tumors derived from injections of CD133(-) cells did not contain any CD133(+) cells, whereas tumors derived from injections of CD133(+) cells did contain CD133(+) cells, suggesting self-renewing capability. However, the proportion of CD133(+) cells in the newly formed tumors in vivo was lower than the proportion of CD133(+) cells in vitro. In conclusion, the human colon cancer cell line, SW620, contains both CD133(+) and CD133(-) phenotypes, and the CD133(+) phenotype has characteristics consistent with those of cancer stem cells.