Polo-like kinase 1 (PLK1) is an important mitotic kinase and its expression is tightly regulated in the cell cycle and in the DNA damage response. PLK1 expression is previously shown to be suppressed by p53 and/or p21. Here, we demonstrate that the CCAAT box in the PLK1 promoter is pivotal for p53/p21-mediated PLK1 repression. Chromatin immunoprecipitation showed that cyclin-dependent kinase 2 (CDK2) associated with the CCAAT box-containing region of PLK1 promoter in unstressed cells, whereas adriamycin (ADR) induced the recruitment of p21 with a concomitant reduction in the occupancy of CDK2 in this region. Expression of p21 inhibited the interaction between CDK2 and the nuclear factor YA (NF-YA) subunit of the CCAAT box-binding transcription factor NF-Y. A mutant p21 that is defective in CDK2 binding was unable to disrupt the CDK2-NF-YA interaction or suppress PLK1 transcription. Co-immunoprecipitation experiments demonstrated the interaction between NF-YA and p21, and in vitro assays showed that p21 could directly bind to NF-YA. Knockdown of NF-YA decreased the amount of PLK1 promoter-associated p21 and abolished p21-mediated PLK1 repression in cells treated with ADR. Depletion of NF-YA diminished the p53-regulated transcriptional activation and suppressed the p53-mediated protection from mitotic death after DNA damage, and these effects of NF-YA deletion were alleviated by PLK1 depletion. Our findings have uncovered a novel p21/NF-YA/PLK1 axis critical for maintaining the checkpoint function of p53 to prevent mitotic death in the DNA damage-induced response.