Separation of clathrin-coated pits from the plasma membrane, a key event during endocytosis, is thought to be driven by dynamin and the actin cytoskeleton. However, the mechanism for the actin-mediated endocytosis remains elusive. RNA interference-mediated suppression of cortactin, an F-actin binding protein that promotes Arp2/3 complex-mediated actin polymerization, effectively blocked transferrin uptake. Depletion of cortactin in brain cytosol inhibited formation of clathrin-coated vesicles by 70% as analyzed in a cell-free system. Interestingly, the interaction between cortactin and dynamin 2 in cells was dependent on actin polymerization and was attenuated upon cell exposure to cytochalasin D as analyzed by immunofluorescence and immunoprecipitation. Moreover, a cortactin mutant deficient in Arp2/3 binding colocalized less efficiently with dynamin 2 and inhibited the uptake of transferrin. The effect of actin polymerization on the interaction between cortactin and the dynamin proline-rich domain (PRD) was further evaluated under a condition for actin polymerization in vitro. Cortactin binds to the dynamin PRD with an equilibrium dissociation constant of 81 nM in the presence of the Arp2/3 complex and actin, and 617 nM in the absence of actin polymerization. Taken together, these data demonstrate that Arp2/3-mediated actin polymerization regulates the accessibility of cortactin to dynamin 2 and imply a novel mechanism by which cortactin and dynamin drive the fission of clathrin-coated pits in an actin polymerization dependent manner.