Aurora kinases play critical roles in chromosome segregation and cell division. Furthermore, Aurora A and B kinases are both frequently amplified and overexpressed in a various kinds of human cancers including leukemia disorders. To address its possibility as a therapeutic target for leukemia, we employed the RNA interference technique to knockdown Aurora kinase gene expression and analyzed its effect on the cell cycle distribution and the chemotherapy-induced cell death in leukemia cells. The expression of Aurora kinase A (AURKA) and Aurora kinase B (AURKB) was observed in all the leukemia cell lines evaluated and 15 out of 18 cultured leukemic cell specimens obtained from untreated patients with acute myeloid leukemia (AML). An arrest in the G(2)/M phase 24 h after specific knockdown of AURKA was shown in U937 leukemia cells. Following the silencing of AURKA, we observed a striking increase in the proportion of cells in the S phase, tetraploid state, and showing multi-nucleated morphologic changes. Cell death was observed in 17.0 ± 0.9% and 20.1 ± 2.5% of U937 cells after AURKA and AURKB silencing, respectively. The proportion of cells in the subG(0)/G(1) phase was 23% and 11% with AURKA and AURKB silencing, respectively. Although the disruption of mitochondrial membrane potential was observed after AURKA or AURKB silencing in U937 cells, the cleavage of caspase-3, -8, -9, and PARP was not observed in the apoptotic cells. Pretreatment of U937 cells with the caspase-3 inhibitor, DEVD-CHO, did not abrogate the AURKA or AURKB silencing-induced cell death. The U937 leukemia cells are resistant to Ara-C-induced cell death. However, we observed a striking synergistic enhancement of the cytotoxicity of Ara-C (10 μM), an important antimetabolic chemotherapeutic agent used for acute leukemia, by the RNA interference-mediated knockdown of AURKA and AURKB through a caspase-independent manner in U937 cells. Collectively, we demonstrate that the specific inhibition of either AURKA or AURKB can potentially regulate the cell cycle distribution of leukemia cells and induce synergistic interaction with Ara-C for inducing the cell death through the mechanism of mitotic catastrophe. These results indicate that Aurora kinase inhibition provides a novel approach for the treatment of human acute leukemia.