The axonal shafts of neurons contain bundled microtubules, whereas extending growth cones contain unbundled microtubule filaments, suggesting that localized activation of microtubule-associated proteins (MAP) at the transition zone may bundle these filaments during axonal growth. Dephosphorylation is thought to lead to MAP activation, but specific molecular pathways have remained elusive. We find that Spinophilin, a Protein-phosphatase 1 (PP1) targeting protein, is responsible for the dephosphorylation of the MAP Doublecortin (Dcx) Ser 297 selectively at the "wrist" of growing axons, leading to activation. Loss of activity at the "wrist" is evident as an impaired microtubule cytoskeleton along the shaft. These findings suggest that spatially restricted adaptor-specific MAP reactivation through dephosphorylation is important in organization of the neuronal cytoskeleton.