Alpha-synuclein (αlphaS) misfolding is associated with Parkinson's disease (PD) but little is known about the mechanisms underlying αS toxicity. Increasing evidence suggests that defects in membrane transport play an important role in neuronal dysfunction. Here we demonstrate that the GTPase Rab8a interacts with αS in rodent brain. NMR spectroscopy reveals that the C-terminus of αlphaS binds to the functionally important switch region as well as the C-terminal tail of Rab8a. In line with a direct Rab8a/αS interaction, Rab8a enhanced αS aggregation and reduced αS-induced cellular toxicity. In addition, Rab8 – the Drosophila ortholog of Rab8a – ameliorated αlphaS-oligomer specific locomotor impairment and neuron loss in fruit flies. In support of the pathogenic relevance of the αlphaS–Rab8a interaction, phosphorylation of αlphaS at S129 enhanced binding to Rab8a, increased formation of insoluble αlpha S aggregates and reduced cellular toxicity. Our study provides novel mechanistic insights into the interplay of the GTPase Rab8a and αalphaS cytotoxicity, and underscores the therapeutic potential of targeting this interaction.