A characteristic pathology of virtually every neurodegenerative disease, including of prion disorders, is the presence of axonal swellings in neurons containing misfolded protein aggregates. These axonopathies also contain accumulations of abnormal amounts of cytoskeletal filaments, microtubule-associated molecular motor proteins, vesicles, and organelles, and occur early in disease, but how they form as well as how they disrupt neuronal viability, are unknown. We have identified a novel mechanism by which formation of pathogenic mutant prion aggregates in axons is a consequence of the targeting of prion-containing vesicles to pre-lysosomal compartments by kinesin-1, a molecular motor required for their formation. Misfolded prions turn on neuronal ER stress, and Golgi-derived mutant prion-containing vesicles engage the ‘rapid ER-stress induced export (RESET)’ secretory/endocytic pathway within axons, to form and maintain prion aggregates. Prion aggregates that form in swellings all along axons are poorly degraded in lysosomes as a result of defective acidification and defective retrograde transport to the soma and thus do not properly degrade, leading to their progressive accumulation and aggregation at distal axonal sites. Super-resolution/electron microscopy studies as well as dynamic live super-resolution imaging show that these aggregates disrupt selectively a subset of post-translationally modified microtubule cytoskeletal tubules at aggregate sites, and thus selectively disrupt the transport of organelles along the axon, including mitochondria. Our data provide a direct relationship between intracellular transport and misfolded protein aggregate formation and demonstrate that formation of prion aggregates in axons is a late event strictly dependent on kinesin-1 motor activity, and that a consequence of aggregate formation is disruption of organelle motility via selective impairments to the axonal cytoskeleton.