The Aurora family of serine/threonine kinases plays important roles in process of cell division or mitosis. Overexpression of Aurora A, B, and C has been identified in many human cancers including colon carcinoma cells. To date, a number of small molecular inhibitors have been developed for reducing Aurora kinases activities of tumor cells in preclinical and clinical trials. In this study, we describe the properties of AM-005, a novel and orally active Aurora A/B kinase inhibitor. AM-005 irreversibly inhibited the proliferation and levels of phospho-Histone H3 in human colon carcinoma cell lines. Defective mitosis was also visualized in AM-005-treated HT29 cells by microscopy. Flow cytometric analysis showed that AM-005 induced the accumulation of HT29 cells with >4N DNA content in a time or concentration-dependent manner, and HT29 cells underwent severe apoptosis at 72 h. Moreover, AM-005 given intragastrically led to suppression of the proliferative response in the xenograft model of colon carcinoma. These results indicate the utility of AM-005 as a promising noncytotoxic agent for treating human colon carcinoma.