In the adult central nervous tissues, axons are incapable of spontaneously regenerating through the lesion site after injury. Regenerating axons display growth cones at their tips and elongate under the normal condition. At injured sites, growth cones are forced to swell and stop. These abnormal growth cones are known as dystrophic endballs. The formation of dystrophic endballs is a hallmark of regeneration failure of axons. It has been well established that an extracellular gradient of proteoglycans at the glial scar is responsible for the dystrophic endball formation. In vitro, the dystrophic endball formation is induced in adult mouse DRG neurons cultured on a gradient of increasing concentration of proteoglycans. Dystrophic endballs contain numerous vacuoles. Molecular mechanisms and intracellular events underlying the dystrophic endball formation remain elusive. Here, we show that autophagy is associated with the dystrophic endball formation by employing an in vitro model of a proteoglycan gradient. Using electron microscopy, we have identified a significant number of vacuoles in dystrophic endballs as autophagosomes, which contain cytosolic fractions and mitochondria. It has been also verified that a number of vacuoles are positive for LC3, a marker of autophagosomes. Furthermore, a treatment of adult mouse DRG neurons with choloroquine or bafilomycin A1, which blocks the fusion of autophagosomes and lysosomes, induced the formation of dystrophic endball-like structures. These data suggested that perturbation in autophagy flux induced by a proteoglycan gradient leads to the dystrophic endball formation.