Background: We have previously reported Signal Transducer and Activator of Transcription 3 (STAT3) to be constitutively activated in aldehyde dehydrogenase (ALDH)(+)/ cluster of differentiation-133 (CD133)(+) colon cancerinitiating cells. In the present study we tested the efficacy of inhibiting STAT3 signaling in human colon cancer-initiating cells by ursolic acid (UA), which exists widely in fruits and herbs. Results: Our data demonstrated that UA inhibited STAT3 phosphorylation, and induced caspase-3 cleavage of ALDH(+)/CD133(+) colon cancer-initiating cells. UA also reduced cell viability and inhibited tumor sphere formation of colon cancer-initiating cells, more potently than two other natural compounds, resveratrol and capsaicin. UA also inhibited the activation of STAT3 induced by interleukin-6 in DLD-1 colon cancer cells. Furthermore, daily administration of UA suppressed HCT116 tumor growth in mice in vivo. Conclusion: Our results suggest STAT3 to be a target for colon cancer prevention. UA, a dietary agent, might offer an effective approach for colorectal carcinoma prevention by inhibiting persistently activated STAT3 in cancer stem cells.