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We have tested whether the anticancer peptide, PNC-27, that kills cancer cells but not normal cells by binding to cancer cell membrane HDM-2 forming pores, kills CD44+ colon cancer stem cells. Flow cytometry determined the CD44 and HDM-2 expression on six-colon cancer cell lines and one normal cell line (CCD-18Co). MTT, LDH release, annexin V binding and caspase 3 assays were used to assess PNC-27-induced cell death. Bioluminescence imaging measured PNC-27 effects on in vivo tumor growth. High percentages of cells in all six tumor lines expressed CD44. PNC-27 co-localized with membrane HDM-2 only in the cancer cells and caused total cell death (tumor cell necrosis, high LDH release, negative annexin V and caspase 3). In vivo, PNC-27 caused necrosis of tumor nodules but not of normal tissue. PNC-27 selectively kills colon cancer stem cells by binding of this peptide to membrane H/MDM-2.