Dopaminergic neurodegeneration in Parkinson's disease (PD) is associated with abnormal dopamine metabolism by MAO-B (monoamine oxidase-B) and intracellular alpha-Synuclein (alpha-Syn) aggregates, called the Lewy body. However, the molecular relationship between alpha-Syn and MAO-B remains unclear. Here, we show that alpha-Syn directly binds to MAO-B and stimulates its enzymatic activity, which triggers AEP (asparagine endopeptidase; legumain) activation and subsequent alpha-Syn cleavage at N103, leading to dopaminergic neurodegeneration. Interestingly, the dopamine metabolite, DOPAL, strongly activates AEP, and the N103 fragment of alpha-Syn binds and activates MAO-B. Accordingly, overexpression of AEP in SNCA transgenic mice elicits alpha-Syn N103 cleavage and accelerates PD pathogenesis, and inhibition of MAO-B by Rasagiline diminishes alpha-Syn-mediated PD pathology and motor dysfunction. Moreover, virally mediated expression of alpha-Syn N103 induces PD pathogenesis in wild-type, but not MAO-B-null mice. Our findings thus support that AEP-mediated cleavage of alpha-Syn at N103 is required for the association and activation of MAO-B, mediating PD pathogenesis.