The DEAD-box RNA helicase DDX3 plays divergent roles in tumorigenesis, however, its function in mitosis is unclear. Immunofluorescence indicated that DDX3 localized to centrosome throughout the cell cycle and colocalized with centrosome-associated p53 during mitosis in HCT116 and U2OS cells. DDX3 depletion promoted chromosome misalignment, segregation defects and multipolar mitosis, eventually leading to G2/M delay and cell death. DDX3 prevented multipolar mitosis by inactivation and coalescence of supernumerary centrosomes. DDX3 silencing suppressed Ser15 phosphorylation of p53 which is required for p53 centrosomal localization. Additionally, knockout of p53 dramatically diminished the association of DDX3 with centrosome, which was rescued by overexpression of the centrosomal targeting-defective p53 S15A mutant, indicating that centrosomal localization of DDX3 is p53 dependent but not through centrosomal location of p53. Furthermore, DDX3 knockdown suppressed p53 transcription through activation of DNA methyltransferases (DNMTs) along with hypermethylation of p53 promoter and promoting the binding of repressive histone marks to p53 promoter. Moreover, DDX3 modulated p53 mRNA translation. Taken together, our study suggests that DDX3 regulates epigenetic transcriptional and translational activation of p53 and colocalizes with p53 at centrosome during mitosis to ensure proper mitotic progression and genome stability, which supports the tumor-suppressive role of DDX3.