The mitotic spindle checkpoint plays a crucial role in regulating accurate chromosome segregation and preventing the adaptation of multiploid progeny cells. Recent reports have indicated that the induction of p53 by mitotic checkpoint activation is essential for protecting cells from abnormal chromosome ploidization caused by mitotic failure. However, although studies have shown that p53 deficiencies arrest mitosis, compromise apoptosis, and may cause profound aneuploidy, the molecular mechanisms leading to p53 induction following mitotic checkpoint activation remain unknown. Here, we show that the BubR1 mitotic checkpoint kinase interacts with p53 both in vitro and in vivo, with higher levels of interaction in mitotic cells. This interaction contributes to p53 phosphorylation. Silencing of BubR1 expression reduces the phosphorylation and stability of p53, whereas exogenous introduction of BubR1 proteins into BubR1-depleted cells recovers p53 stability. In addition, inhibition of BubR1 expression in the presence of a microtubule inhibitor accelerates chromosomal instability and polyploidy in p53-null cells. These results collectively suggest that p53 activation in response to mitotic spindle damage requires signaling via BubR1-mediated phosphorylation.