Mitotic kinases regulate cell division and its checkpoints, errors of which can lead to aneuploidy or genetic instability. One of these is Aurora-B, a key kinase that is required for chromosome alignment at the metaphase plate and for cytokinesis in mammalian cells. We report here that human Aurora-B is phosphorylated at Thr-232 through interaction with the inner centromere protein (INCENP) in vivo. The phosphorylation of Thr-232 occurs by means of an autophosphorylation mechanism, which is indispensable for the Aurora-B kinase activity. The activation of Aurora-B spatio-temporally correlated with the site-specific phosphorylation of its physiological substrates, histone H3 and vimentin. Overexpression of the TA mutant of Aurora-B, in which Thr-232 was changed into alanine, frequently induced multinuclearity in cells. These results indicate that the phosphorylation of Thr-232 is an essential regulatory mechanism for Aurora-B activation.