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The microtubule-dependent molecular motor KIF23 (Kinesin family member 23) is one of two components of the centralspindlin complex assembled during late stages of mitosis. Formation of this complex is known as an essential step for cytokinesis. Here, we identified KIF23 as a new transcriptional target gene of the tumor suppressor protein p53. We showed that p53 reduces expression of KIF23 on the mRNA as well as the protein level in different cell types. Promoter reporter assays revealed that this repression results from downregulation of KIF23 promoter activity. CDK inhibitor p21(WAF1/CIP1) was shown to be necessary to mediate p53-dependent repression. Furthermore, we identified the highly conserved cell cycle genes homology region (CHR) in the KIF23 promoter to be strictly required for p53-dependent repression as well as for cell cycle-dependent expression of KIF23. Cell cycle- and p53-dependent regulation of KIF23 appeared to be controlled by differential binding of DREAM and MMB complexes to the CHR element. With this study, we describe a new mechanism for transcriptional regulation of KIF23. Considering the strongly supporting function of KIF23 in cytokinesis, its p53-dependent repression may contribute to the prevention of uncontrolled cell growth.