The abnormal aggregation of fibrillar alpha-synuclein in Lewy bodies plays a critical role in the pathogenesis of Parkinson's disease. However, the molecular mechanisms regulating alpha-synuclein pathological effects are incompletely understood. Here we show that alpha-synuclein binds phosphoinositide-3 kinase enhancer L (PIKE-L) in a phosphorylation-dependent manner and sequesters it in Lewy bodies, leading to dopaminergic cell death via AMP-activated protein kinase (AMPK) hyperactivation. alpha-Synuclein interacts with PIKE-L, an AMPK inhibitory binding partner, and this action is increased by S129 phosphorylation through AMPK and is decreased by Y125 phosphorylation via Src family kinase Fyn. A pleckstrin homology (PH) domain in PIKE-L directly binds alpha-synuclein and antagonizes its aggregation. Accordingly, PIKE-L overexpression decreases dopaminergic cell death elicited by 1-methyl-4-phenylpyridinium (MPP+), whereas PIKE-L knockdown elevates alpha-synuclein oligomerization and cell death. The overexpression of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) or alpha-synuclein induces greater dopaminergic cell loss and more severe motor defects in PIKE-KO and Fyn-KO mice than in wild-type mice, and these effects are attenuated by the expression of dominant-negative AMPK. Hence, our findings demonstrate that alpha-synuclein neutralizes PIKE-L's neuroprotective actions in synucleinopathies, triggering dopaminergic neuronal death by hyperactivating AMPK.