Salinomycin is a monocarboxylic polyether antibiotic that has been reported to induce apoptosis in various types of cancer cells with specificity for cancer stem cells. However, its anticancer effect in colorectal cancer stem cells has never been reported. In the present study, we examined the ability of salinomycin to induce cell death in the colorectal cancer stem cell line CD44+EpCAM+ HCT-116, and we measured its in vivo tumor inhibition capacity. Salinomycin dose-dependently induced cytotoxicity in the CD44+EpCAM+ HCT-116 cells and inhibited colony formation. Salinomycin treatment was shown to induce apoptosis, as evidenced by nuclear fragmentation, an increase in the proportion of acridine orange/ethidium bromide-positive cells and an increase in the percentage of Annexin V-positive cells. Apoptosis was induced in colorectal cancer stem cells in a caspase-dependent manner, as shown by an increase in the levels of cleaved caspase-3, -8 and -9. JC-1 staining further revealed that salinomycin induced colorectal cancer cell apoptosis via the mitochondrial pathway. In addition, salinomycin treatment of xenograft mice inhibited the growth of tumors derived from the CD44+EpCAM+ HCT-116 cells. The present study demonstrated that the antibiotic salinomycin exerts an anti-colorectal cancer effect in vitro and in vivo, suggesting salinomycin as a potential drug for colorectal cancer therapy.