Parkinson's disease (PD) is pathologically characterized by selective loss of dopaminergic neurons in the midbrain and the existence of intracellular protein inclusions termed Lewy bodies, largely composed of α-synuclein. Genetic studies have revealed that rare point mutations in the gene encoding α-synuclein including A30P, A53T, and E46K are associated with familial forms of PD, indicating a pathological role for mutant α-synuclein in PD etiology. However, the mechanisms underlying the neuronal toxicity of mutant α-synuclein are still to be elucidated. Growing evidence has suggested a deleterious effect of mutant α-synuclein on the autophagy-lysosome pathway. In this study, we discovered that overexpression of human E46K mutant α-synuclein impaired macroautophagy in mammalian cells. Our data showed that overexpression of E46K mutant α-synuclein impaired autophagy at an early stage of autophagosome formation via the c-Jun N-terminal kinase 1 (JNK1)-Bcl-2 but not the mammalian target of rapamycin (mTOR) pathway. Overexpressed E46K mutant α-synuclein inhibited JNK1 activation, leading to a reduced Bcl-2 phosphorylation and increased association between Bcl-2 and Beclin1, further disrupting the formation of Beclin1/hVps34 complex, which is essential for autophagy initiation. Furthermore, overexpression of E46K mutant α-synuclein increased the vulnerability of differentiated PC12 cells to rotenone treatment, which would be partly due to its inhibitory effects on autophagy. Our findings may shed light on the potential roles of mutant α-synuclein in the pathogenesis of PD.