The regulation of protein tyrosine phosphorylation is an important aspect during the cell cycle. From G2-M transition to mitotic anaphase, phosphorylation of Tyr421, Tyr466 and Tyr482 of cortactin, an actin-filament associated protein, is dramatically induced. The phosphorylated cortactin is almost exclusively associated with centrosomes or spindle poles during mitosis. At G2-M transition prior to the breakdown of the nuclear envelope, two duplicated centrosomes migrate towards opposite ends of the nucleus to form the spindle poles. This centrosome-separation process and also the start of mitosis are inhibited or delayed by the depolymerization of actin filaments. Also inhibited is the separation of centrosomes when a truncated form of cortactin is expressed, whose C-terminus contains the tyrosine phosphorylation region but lacks the actin-binding domains. We introduced mutations at the tyrosine phosphorylation sites in the truncated C-terminus of cortactin and found that the C-terminus could no longer interfere with centrosome separation process. Our study shows that, cortactin phosphorylated at Tyr421, Tyr466 and Tyr482 mediates the actin-filament-driven centrosome separation at G2-M transition by providing a bridge between the centrosome and actin-filaments.