STAT3 is constitutively activated in colon cancer but its contributions in cancer-initiating cells have not been explored. In this study, we characterized STAT3 in aldehyde dehydrogenase (ALDH)-positive (ALDH⁺) and CD133-positive (CD133⁺) subpopulations of human colon tumor cells that exhibited more potent tumor-initiating ability than ALDH⁻/CD133⁻ cells in tumor xenograft assays in mice. We found that ALDH⁺/CD133⁺ cells expressed higher levels of the active phosphorylated form of STAT3 than either ALDH⁻/CD133⁻ or unfractionated colon cancer cells. STAT3 inhibition by RNA interference–mediated knockdown or small-molecule inhibitors LLL12 or Stattic blocked downstream target gene expression, cell viability, and tumorsphere-forming capacity in cancer-initiating cells. Similarly, treatment of mouse tumor xenografts with STAT3 short hairpin RNA (shRNA), interleukin 6 shRNA, or LLL12 inhibited tumor growth. Our results establish that STAT3 is constitutively activated in colon cancer–initiating cells and that these cells are sensitive to STAT3 inhibition. These findings establish a powerful rationale to develop STAT3 inhibitory strategies for treating advanced colorectal cancers. Cancer Res; 71(23); 7226–37. ©2011 AACR.