Aurora kinases, frequently detected to be over-expressing in human tumors, regulate many essential events during mitosis progression and have been regarded as potentially important targets for cancer therapy. S39 is a novel potent inhibitor of Aurora B kinase with the IC50 90.07 nM in the biochemical assay in an ATP competitive manner. S39 treatment on human tumor cells can inhibit the phosphorylation of Histone H3 (Ser10), a direct downstream substrate of Aurora B kinase, indicating S39 inhibits endogenous Aurora B kinase activity in cell-based level. Furthermore, S39 treatment blocks cell proliferation, inhibits colony formation and induces apoptosis in a wide range of human tumor cell lines. These results indicate that S39 is a potential lead compound to be an Aurora B inhibitor.