The accumulation of autophagosomes within axons is often observed in axonopathies associated with various neurological disorders, including those following excitotoxic insults. Nevertheless, the life cycle of autophagosomes in axons is not well understood. In the present study, we used microexplant cultures of cerebellar granule cells from GFP-LC3 transgenic mice to perform time-lapse imaging of LC3-positive dots in identified axons. Since these GFP-LC3 dots were never observed in granule cells on an Atg5-null background, they were considered to represent autophagosomes. Under physiological conditions, the autophagosomes showed bidirectional and saltatory movement with a bias towards one direction. Such vectorial movement was largely blocked by the dynein motor inhibitor EHNA (erythro-9-[3-(2-hydroxynonyl)] adenine), suggesting that the autophagosomes moved towards the soma, where most lysosomes are located. Interestingly, the application of the glutamate analog Ν-methyl-D-aspartic acid (NMDA) as an excitotoxin increased the number of autophagosomes in axons, while it did not significantly change its movement characteristics. These results suggest that autophagosomes play important roles in axons and are dynamically regulated under physiological and pathological conditions.