The extreme polarized morphology of neurons poses a challenging problem for intracellular trafficking pathways. The distant synaptic terminals must communicate via axonal transport with the cell soma for neuronal survival, function, and repair. Multiple classes of organelles transported along axons may establish and maintain the polarized morphology of neurons, as well as control signaling and neuronal responses to extracellular cues such as neurotrophic or stress factors.Wereported previously that the motor-binding protein Sunday Driver (syd), also known as JIP3 or JSAP1, links vesicular axonal transport to injury signaling. To better understand syd function in axonal transport and in the response of neurons to injury, we developed a purification strategy based on anti-syd antibodies conjugated to magnetic beads to identify syd-associated axonal vesicles. Electron microscopy analyses revealed two classes of syd-associated vesicles of distinct morphology. To identify the molecular anatomy of syd vesicles, we determined their protein composition by mass spectrometry. Gene Ontology analyses of each vesicle protein content revealed their unique identity and indicated that one class of syd vesicles belongs to the endocytic pathway, whereas another may belong to an anterogradely transported vesicle pool. To validate these findings, we examined the transport and localization of components of syd vesicles within axons of mouse sciatic nerve. Together, our results lead us to propose that endocytic syd vesicles function in part to carry injury signals back to the cell body, whereas anterograde syd vesicles may play a role in axonal outgrowth and guidance.