Cancer stem cells(cancer initiating cells) have become increasingly important in the treatment of malignant tumors. CD44 in particular has been identified as a marker for stem cells in colon cancer, which is a high-morbidity tumor. However, many details remain unknown, including identification of the relevant exon. The elucidation of these details could lead to the development of new therapies and improvements in prognosis. We report our findings on the importance of CD44 variant exon 9(v9) of stem cells in colon cancer. Using the anti-CD44 standard form(s) antibody, as well as antibodies for each of the CD44 variant exons, we studied colon cancer cell lines by examining stained images of stem cells in the crypt of normal colon mucosa. Using the anti-CD44v9 antibody that fits the normal colon mucosa stem cells, we screened cells using flow cytometry to examine colony formation, resistance to anticancer drugs, and tumor mass formation after subcutaneous implantation in mice. The stem cell-containing region in the crypt of normal colon mucosa was negative for anti-Ki67 antibody staining; only the anti-CD44 v9 antibody stain was expressed. As for colony formation, resistance to anticancer drugs, and tumor mass formation, cells positive both for anti-CD44s and anti-CD44v9 antibody stains was significantly more frequent than those positive for anti-CD44s antibody stain and negative for anti-CD44v9 antibody stain and those negative both for anti-CD44s and anti-CD44v9 antibody stains. CD44 variant exon 9 plays an important role in colon cancer stem cells.