Recent studies have suggested the existence of a small subset of cancer cells called cancer stem cells (CSCs), which possess the ability to initiate malignancies, promote tumor formation, drive metastasis, and evade conventional chemotherapies. Elucidation of the specific signaling pathway and mechanism underlying the action of CSCs might improve the efficacy of cancer treatments. In this study, we analyzed differentially expressed proteins between glioma stem cells and differentiated tumor cells isolated from the human glioma cell line, U251, via iTRAQ-tagging combined with two dimensional liquid chromatography tandem MS analysis to identify proteins correlated with specific features of CSCs. Out of a total data set of 559 identified proteins, 29 proteins were up-regulated in the glioma stem cells when compared with the differentiated cells. Interestingly, The expression level of S100A9 was fivefold higher in glioma stem cells than differentiated cells. Similar results were also observed in glioma stem cells derived from other glioma cells. More importantly, knockdown of S100A9 by RNA interference suppressed the proliferation of glioma stem cell line and decreased the growth of xenograft tumors in vivo. Taken together, these results indicate that the tumorigenesis potential of CSCs arises from highly expressed S100A9. J. Cell. Biochem. 114: 2795–2808, 2013. © 2013 Wiley Periodicals, Inc.