Collapsin response mediator protein 2 (CRMP-2) enhances the advance of growth cones by regulating microtubule assembly and Numb-mediated endocytosis. We previously showed that Rho kinase phosphorylates CRMP-2 during growth cone collapse; however, the roles of phosphorylated CRMP-2 in growth cone collapse remain to be clarified. Here, we report that CRMP-2 phosphorylation by Rho kinase cancels the binding activity to the tubulin dimer, microtubules, or Numb. CRMP-2 binds to actin, but its binding is not affected by phosphorylation. Electron microscopy revealed that CRMP-2 localizes on microtubules, clathrin-coated pits, and actin filaments in dorsal root ganglion neuron growth cones, while phosphorylated CRMP-2 localizes only on actin filaments. The phosphomimic mutant of CRMP-2 has a weakened ability to enhance neurite elongation. Furthermore, ephrin-A5 induces phosphorylation of CRMP-2 via Rho kinase during growth cone collapse. Taken together, these results suggest that Rho kinase phosphorylates CRMP-2, and inactivates the ability of CRMP-2 to promote microtubule assembly and Numb-mediated endocytosis, during growth cone collapse.