Transformation of a transected axonal tip into a growth cone (GC) is a critical step in the cascade leading to neuronal regeneration. Critical to the regrowth is the supply and concentration of vesicles at restricted sites along the cut axon. The mechanisms underlying these processes are largely unknown. Using online confocal imaging of transected, cultured Aplysia californica neurons, we report that axotomy leads to reorientation of the microtubule (MT) polarities and formation of two distinct MT-based vesicle traps at the cut axonal end. Approximately 100 μm proximal to the cut end, a selective trap for anterogradely transported vesicles is formed, which is the plus end trap. Distally, a minus end trap is formed that exclusively captures retrogradely transported vesicles. The concentration of anterogradely transported vesicles in the former trap optimizes the formation of a GC after axotomy.