Colon cancer is one of the most lethal and common malignancies worldwide. STK899704, a novel synthetic agent, has been reported to exhibit anticancer effects towards numerous cancer cells. However, the effect of STK899704 on the biological properties of colon cancer, including cancer cell migration and cancer stem cells (CSCs), remains unknown. Here, we examined the inhibitory effect of STK899704 on cell migration and CSC stemness. In the wound healing assay, STK899704 significantly inhibited the motility of colon cancer cells. Furthermore, STK899704 downregulated the mRNA expression levels of the cell migration mediator focal adhesion kinase (FAK). STK899704 also suppressed mitogen-activated protein kinase kinase and extracellular signal-regulated kinase, which are downstream signaling molecules of FAK. Additionally, STK899704 inhibited stemness gene expression and sphere formation in colon cancer stem cells. These results suggest that STK899704 can be used to treat human colon cancer. [BMB Reports 2018; 51(11): 596-601]