There is growing evidence supporting a role of extracellular alpha-synuclein in the spreading of Parkinson's disease (PD) pathology. Recent pathological studies have raised the possibility that the enteric nervous system (ENS) is one of the initial sites of alpha-synuclein pathology in PD. We therefore undertook this survey to determine whether alpha-synuclein can be secreted by enteric neurons. Alpha-synuclein secretion was assessed by immunoblot analysis of the culture medium from primary culture of ENS. We show that alpha-synuclein is physiologically secreted by enteric neurons via a conventional, endoplasmic reticulum/Golgi-dependent exocytosis, in a neuronal activity-regulated manner. Our study is the first to evidence that enteric neurons are capable of secreting alpha-synuclein, thereby providing new insights into the role of the ENS in the pathophysiology of PD.