Norcantharidin has been used as an efficacious anticancer drug in China for many years, but its true mechanism remains poorly understood. Intriguingly, in an in vitro series study of anticancer drugs, we found that norcantharidin can effectively inhibit epithelial tumor cells from expressing integrin αvβ6. Our previous studies have confirmed that integrin αvβ6 is closely relevant to malignant epithelial cell tumor biology behavior, and it can promote cancer cells to invade and metastasize through a special αvβ6–extracellular signal-related kinase (ERK) direct signaling pathway. In this study, we investigated the relationship between the norcantharidin anticancer mechanism and integrin αvβ6. After HT-29 colon cancer cells were treated with norcantharidin,cell apoptosis increased remarkably. The expression of αvβ6 and the amount of p-ERK decreased substantially; simultaneously, the linkage between αvβ6 and ERK was barely detectable. However, the expression of other integrins and the levels of mitogen-activated protein kinase hardly changed. On these grounds, we presumed that norcantharidin induced HT-29 colon cancer cell apoptosis through the αvβ6–ERK signaling pathway. This finding elicited a novel strategy for targeting the whole αvβ6–ERK signal pathway, rather than simply blocking the combining site of αvβ6–ERK in colon cancer treatment.