Colorectal cancers are the third most common types of cancers worldwide. Surgical resection is unable to eliminate tumors completely due to metastasis. A demand for new chemotherapeutic tools exists. In the present study, we examined the chemopreventive potential of cucurbitacin I, a natural component extracted from plants of the Cucurbitaceae family, in the colon cancer cell line COLO205. We hypothesized that cucurbitacin I would prevent colon cancer cell migration and invasion, and sensitize colon cancer cells to chemotherapy. Our data demonstrated that exposure of the COLO205 cells to cucurbitacin I significantly decreased cell viability. Furthermore our data demonstrated for the first time that in the COLO205 cells, cucurbitacin I could suppress the cell migration and invasion, and harbor chemosensitization activity against colon cancer. The anticancer activity of cucurbitacin I was accomplished by downregulating p-STAT3 and MMP-9 expression. Collectively, our results suggest that cucurbitacin I may be a potent adjuvant chemotherapeutic agent for colon cancer with anti-migration, anti-invasion and chemosensitizing activities.