Cancer stem cells (CSCs) are responsible for cancer formation, recurrence and drug resistance. c‑Myc, one of the core markers for stem cells, has recently been considered to serve as a link between malignancy and ‘stemness’. However, the precise function of c‑Myc in colon CSCs is still unclear. In the present study, a subpopulation of colon CSCs expressing a CD133 surface phenotype was isolated from the human HT‑29 cell line, which possess greater tumor sphere‑forming efficiency and have higher expression of ‘stemness’‑associated genes compared with CD133‑negative cells. Furthermore, it was demonstrated that c‑Myc was highly expressed in CD133+ colon CSCs. Knockdown of c‑Myc expression with small interfering RNA in colon CSCs can significantly inhibit tumor sphere formation, reduce the invasive and migratory capacity of CD133+ cells in vitro, and suppress the tumorigenicity of colon CSCs in vivo. In addition, it was suggested that c‑Myc silencing may sensitize colon CSCs to chemotherapy‑induced cytotoxicity via the downregulation of ABCG2 and ABCB5. These findings support a central role for c‑Myc in maintaining the self‑renewing and chemoresistant properties of colon CSCs.