Aggregated forms of alpha-synuclein play a crucial role in the pathogenesis of synucleinopathies such as Parkinson's disease (PD). However, the molecular mechanisms underlying the pathogenic effects of alpha-synuclein are not completely understood. Here we show that asparagine endopeptidase (AEP) cleaves human alpha-synuclein, triggers its aggregation and escalates its neurotoxicity, thus leading to dopaminergic neuronal loss and motor impairments in a mouse model. AEP is activated and cleaves human alpha-synuclein at N103 in an age-dependent manner. AEP is highly activated in human brains with PD, and it fragments alpha-synuclein, which is found aggregated in Lewy bodies. Overexpression of the AEP-cleaved alpha-synuclein(1-103) fragment in the substantia nigra induces both dopaminergic neuronal loss and movement defects in mice. In contrast, inhibition of AEP-mediated cleavage of alpha-synuclein (wild type and A53T mutant) diminishes alpha-synuclein's pathologic effects. Together, these findings support AEP's role as a key mediator of alpha-synuclein-related etiopathological effects in PD.