Colon cancer is one of the most common cancers worldwide with high mortality. A major issue in colon cancer treatment is drug‐resistance and metastasis that have been ascribed to the cancer stem cells. In this study, we isolated colon cancer stem cells through sphere culture and verified with the cancer stem cell markers CD133, CD44, and CD24. We demonstrated that the PI3K/Akt/mTOR signalling pathway was highly activated in the colon cancer stem cells and that inhibition of the PI3K/Akt/mTOR pathway by the inhibitor BEZ235 suppressed the colon cancer stem cell proliferation with reduced stemness indicated by CD133 and Lgr5 expressions. Treatment with insulin as a known activator of the PI3K/Akt pathway increased CD133 expression and decreased the effects of BEZ235 on colon cancer proliferation and survival. Our data collectively suggest that the PI3K/Akt/mTOR pathway underpins the stemness of colon cancer stem cells and BEZ235 is a good drug candidate potentially for treatment of colon cancer drug resistance and metastasis. This article is protected by copyright. All rights reserved.