CDC25B is one of the three human dual-specificity phosphatases involved in the activation of cyclin-dependent kinases at key stages of the cell division cycle. CDC25B that is responsible for the activation of CDK1-cyclin B1 is regulated by phosphorylation. The STK15/Aurora-A kinase locally phosphorylates CDC25B on serine 353 at the centrosome during the G2/M transition. Here we have investigated this phosphorylation event during the cell cycle, and in response to activation of the G2 DNA damage checkpoint. We show that accumulation of the S353-phosphorylated form of CDC25B at the centrosome correlates with the relocalization of cyclin B1 to the nucleus and the activation of CDK1 at entry into mitosis. Upon activation of the G2/M checkpoint by DNA damage, we demonstrate that Aurora-A is not activated and consequently CDC25B is not phosphorylated. We show that ectopic expression of Aurora-A results in a bypass of the checkpoint that was partially overcome by a S353A mutant of CDC25B. Finally, we show that bypass of the G2/M checkpoint by the CHK1 kinase inhibitor UCN-01 results in the activation of Aurora-A and phosphorylation of CDC25B on S353. These results strongly suggest that Aurora-A-mediated phosphorylation of CDC25B at the centrosome is an important step contributing to the earliest events inducing mitosis, upstream of CDK1-cyclin B1 activation.