Chromosome segregation in mitosis is orchestrated by dynamic interactions between spindle microtubules and centromeres, which in turn are governed by protein kinase- and phosphatase-signaling cascades. Previous studies showed that overexpression of human phosphatase HsCdc14A, an antagonist of cyclin-dependent kinase 1, affects several aspects of cell division. However, the molecular mechanism underlying HsCdc14A regulation in mitosis has remained elusive. Here we show that HsCdc14A activity is regulated by an auto-inhibitory mechanism via its intra-molecular association. Our biochemical study demonstrated that Polo-like kinase 1 (PLK1) interacts with and phosphorylates HsCdc14A. This phosphorylation partially releases the auto-inhibition of HsCdc14A judged by its phosphatase activity in vitro. To examine the functional relevance of such phospho-regulation of HsCdc14A in vivo, a phospho-mimicking mutant of HsCdc14A was expressed in HeLa cells. Importantly, overexpression of the phospho-mimicking mutants caused aberrant chromosome alignment with a prometaphase delay, suggesting the temporal regulation of HsCdc14A activity is critical for orchestrating mitotic events. Given the fact that HsCdc14A forms an intra-molecular association and PLK1-mediated phospho-regulation promotes HsCdc14A phosphatase activity, we propose that PLK1-HsCdc14A interaction provides a temporal regulation of HsCdc14A in chromosome segregation during mitosis.