Heterochromatin protein 1 (HP1) plays an important role in heterochromatin formation and undergoes large-scale, progressive dissociation from heterochromatin in prophase cells. However, the mechanisms regulating the dynamic behavior of HP1 are poorly understood. In this study, the role of Aurora-B was investigated with respect to the dynamic behavior of HP1alpha. Mammalian Aurora-B, AIM-1, colocalizes with HP1alpha to the heterochromatin in G2. Depletion of Aurora-B/AIM-1 inhibited dissociation of HP1alpha from the chromosome arms at the G2-M transition. In addition, depletion of INCENP led to aberrant cellular localization of Aurora-B/AIM-1, but it did not affect heterochromatin targeting of HP1alpha. It was proposed in the binary switch hypothesis that phosphorylation of histone H3 at Ser-10 negatively regulates the binding of HP1alpha to the adjacent methylated Lys-9. However, Aurora-B/AIM-1-mediated phosphorylation of H3 induced dissociation of the HP1alpha chromodomain but not of the intact protein in vitro, indicating that the center and/or C-terminal domain of HP1alpha interferes with the effect of H3 phosphorylation on HP1alpha dissociation. Interestingly, Lys-9 methyltransferase SUV39H1 is abnormally localized together along the metaphase chromosome arms in Aurora-B/AIM-1-depleted cells. In conclusion, these results showed that Aurora-B/AIM-1 is necessary for regulated histone modifications involved in binding of HP1alpha by the N terminus of histone H3 during mitosis.