Human α-synuclein overexpression and its toxic accumulation in neurons or glia are known to play key roles in the pathogenesis of Parkinson's disease and other related neurodegenerative synucleinopathies. Several single point mutations in the α-synuclein gene, as well as gene duplication and triplication, have been linked to familial Parkinson's disease. Moreover, genetic variability of the α-synuclein gene promoter is associated with idiopathic Parkinson's disease. Silencing of the human α-synuclein gene by vector-based RNA interference (RNAi) is a promising therapeutic approach for synucleinopathies. Here, we report identification of a 21-nucleotide sequence in the coding region of human α-synuclein that constitutes an effective target for robust silencing by RNAi and demonstrate allele-specific silencing of the A53T mutant of human α-synuclein. Furthermore, we have developed a plasmid vector-based RNAi for silencing of human α-synuclein in vitro. Lastly, using a dual cassette lentivirus that co-expresses an α-synuclein-targeting small hairpin RNA (shRNA) and enhanced green fluorescent protein (EGFP) as a marker gene, we demonstrate effective silencing of endogenous human α-synuclein in vitro in the human dopaminergic cell line SH-SY5Y and also of experimentally expressed human α-synuclein in vivo in rat brain. Our results demonstrate potent silencing of human α-synuclein expression in vitro and in vivo by viral vector-based RNAi and provide the tools for developing effective gene silencing therapeutics for synucleinopathies, including Parkinson's disease.