Aurora-B is a protein kinase required for chromosome segregation and the progression of cytokinesis during the cell cycle. We report here that Aurora-B phosphorylates GFAP and desmin in vitro, and this phosphorylation leads to a reduction in filament forming ability. The sites phosphorylated by Aurora-B; Thr-7/Ser-13/Ser-38 of GFAP, and Thr-16 of desmin are common with those related to Rho-associated kinase (Rho-kinase), which has been reported to phosphorylate GFAP and desmin at cleavage furrow during cytokinesis. We identified Ser-59 of desmin to be a specific site phosphorylated by Aurora-B in vitro. Use of an antibody that specifically recognized desmin phosphorylated at Ser-59 led to the finding that the site is also phosphorylated specifically at the cleavage furrow during cytokinesis in Saos-2 cells. Desmin mutants, in which in vitro phosphorylation sites by Aurora-B and/or Rho-kinase are changed to Ala or Gly, cause dramatic defects in filament separation between daughter cells in cytokinesis. The results presented here suggest the possibility that Aurora-B may regulate cleavage furrow-specific phosphorylation and segregation of type III IFs coordinatedly with Rho-kinase during cytokinesis.