Glycans exhibit characteristic changes in their structures during development and thus have been used as markers for stem/progenitor cells. However, the glycan structures unique to cancer stem cells (CSC) remain unknown. In the present study, we examined glycan structures in CD133+CD13+ CSC, which were recently found to have a high CSC ability, by means of a lectin microarray. Seven sialylated glycan‐recognizing lectins, MAL‐I, SNA, SSA, TJA‐I, ACG, ABA and MAH, showed higher affinity to CD133+CD13+ CSC than CD133+ cells with a lower CSC ability. In addition, we demonstrated that CD133+SSA+ cells isolated from Huh7 cells had a significantly higher ability to form tumors in non‐obese diabetic/severe combined immunodeficiency disease (NOD/SCID) mice and spheres under serum‐free conditions than CD133+SSA− cells. These results suggest that hepatic CSC highly express sialylated glycans and that SSA lectin can be used as a tool for isolating CSC. This study is the first report to demonstrate the characteristic glycan structures in CSC and to indicate a new methodology involving lectins for isolating CSC. (Cancer Sci 2011; 102: 1164–1170)