The tumor suppressor kinases LATS1 and LATS2 (LATS1/2) regulate not only organ size through the Hippo signaling pathway， but also cell-cycle checkpoints and apoptosis via other signaling cascades. We previously reported that LATS1/2 localize to the mitotic apparatus， where they are involved in the phosphorylation and activation of the mitotic kinase Aurora-B; however， the detailed mechanism of LATS1/2 action remains obscure. The activity of Aurora-B is stringently regulated by formation of the chromosomal passenger complex containing the inner centromere protein (INCENP)， which leads to appropriate activation of Aurora-B during mitosis and cytokinesis. In this study， we found that LATS1/2 phosphorylated INCENP at S894 in the Thr-Ser-Ser motif. Moreover， the LATS-mediated phosphorylation of S894 was necessary and sufficient for the activation of Aurora-B， which is required for completion of cytokinesis in cells engaged in multipolar division. We propose a novel mechanism for regulation of Aurora-B via INCENP phosphorylation by LATS1/2 during cytokinesis.