Cancer stem cells (CSCs) are resistant to radiotherapy and chemotherapy and play a significant role in cancer recurrence. Design of better treatment strategies that can eliminate or otherwise control CSC populations in tumors is necessary. In this study, the sensitivity to tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced cytotoxicity and the effect of amurensin G, a novel sirtuin 1 (SIRT1) inhibitor, were examined using the CSC-enriched fraction of HCT-15 human colon cancer cells. Cancer stem cell-enriched HCT-15 colony cells were paradoxically less sensitive to doxorubicin, and more sensitive to TRAIL-induced cytotoxicity, than their parental cells. Also, CD44+ HCT-15 cells were more susceptible to TRAIL-mediated cytotoxicity than CD44 HCT-15 cells, possibly due to increased levels of death receptors DR4 and DR5 as well as c-Myc, and decreased levels of c-FLIPL/S in CD44+ cells compared with CD44 HCT-15 cells. The combination effect of amurensin G on TRAIL-mediated cytotoxicity was much more apparent in CD44+ cells than in CD44 HCT-15 cells, and this was associated with more prominent downregulation of c-FLIPL/S in CD44+ cells than in CD44 HCT-15 cells. These results indicate that HCT-15 colony or CD44+ cells, which may have CSC properties, are more sensitive to TRAIL than parental or CD44 HCT-15 cells. Amurensin G may be effective in eliminating colon CSCs and be applicable to potentiate the sensitivity of colon CSCs to TRAIL.