The p53 tumor suppressor inhibits the proliferation of cells which undergo prolonged activation of the mitotic checkpoint. However, the function of this antiproliferative response is not well defined. Here we report that p53 suppresses structural chromosome instability following mitotic arrest in human cells. In both HCT116 colon cancer cells and normal human fibroblasts, DNA breaks occurred during mitotic arrest in a p53-independent manner, but p53 was required to suppress the proliferation and structural chromosome instability of the resulting polyploid cells. In contrast, cells made polyploid without mitotic arrest exhibited neither significant structural chromosome instability nor p53-dependent cell cycle arrest. We also observed that p53 suppressed both the frequency and structural chromosome instability of spontaneous polyploids in HCT116 cells. Furthermore, time-lapse videomicroscopy revealed that polyploidization of p53−/− HCT116 cells is frequently accompanied by mitotic arrest. These data suggest that a function of the p53-dependent postmitotic response is the prevention of structural chromosome instability following prolonged activation of the mitotic checkpoint. Accordingly, our study suggests a novel mechanism of tumor suppression for p53, as well as a potential role for p53 in the outcome of antimitotic chemotherapy.