Neurons have a distinct structure; they are the only cells in the body whose proximal and distal ends can be separated by more than a meter. This leads to unique challenges for the neuron, specifically, how proteins, organelles, and other cargo synthesized in the cell body are specifically trafficked from the soma to the distal end of the axon, and how degradative cargo and signaling factors are transported from the terminal to the cell body. Using primary cultured central and peripheral neurons isolated from the brains and spinal cords of rats or mice, we sought to determine if a neuronal-specific kinase, cyclin dependent kinase 5 (CDK5) regulates the motility of cargo moving along the axon, and if this kinase also regulates the localized exclusion of somatodendritic cargo from the axon. Within the mid-axon, we observed that baseline CDK5 activity was not required to regulate axonal transport, but pathological activation of CDK5 via a stress-associated activator disrupted both anterograde (outward) and retrograde (inward) motility. In contrast, within the axon initial segment (AIS), inhibition of normal CDK5 activity disrupted cytoskeletal structure and compromised axonal and dendritic sorting, aberrantly permitting the entry of somatodendritic cargos into the axon. We determined that both roles of CDK5 in axonal regulation were dependent on phosphorylation of target Ndel1, a protein that regulates the interaction of the retrograde microtubule-based motor dynein with its cofactor Lis1. In the mid-axon, high levels of CDK5 activity causes dynein to tightly bind along the microtubule interrupting processive motility, while in the AIS, CDK5 activity is required to initiate dynein-driven return of somatodendritic cargo to the cell body. Together theses studies demonstrate the importance of CDK5 activity in the regulation of transport within the neuron.