Alpha-synuclein (alpha-Syn) and phosphatase and tensin homolog deleted on chromosome ten (PTEN)-induced putative kinase (PINK) 1 are proteins found in Lewy bodies, which are a pathological hallmark of Parkinson's disease (PD). PINK1 overexpression suppresses alpha-Syn-induced phenotypes and increases lifespan and health in an animal model of PD. It has been suggested that the two proteins regulate protein phosphatase (PP) 2A activity, but the underlying mechanisms and neuroprotective action of PP2A against PD-associated pathology are unknown. We found that alpha-Syn overexpression in SK-N-SH neuroblastoma cells and primary cortical neurons caused mitochondrial dysfunction and cell injury via phosphorylation of PP2A at Tyr307 and inhibition of its activity. Concomitant overexpression of PINK1 reversed this effect and restored the activity. The level of phospho-activated Src was increased in cells overexpressing alpha-Syn, which was reversed by co-expressing PINK1, suggesting that the latter suppressed a-Syn-induced PP2A inactivation by inhibiting Src activity. Calmodulin/Src complex formation was also enhanced in alpha-Synoverexpressing cells, which was reversed by co-expression of PINK1 as a result of reduced mitochondrial Ca2+ releasing. Interestingly, the protective effects of PINK1 in alpha-Syn induced models were abolished by treatment with the PP2A inhibitor okadaic acid, indicating that PP2A is a target of PINK1. These findings indicate that PINK1 protects against alpha-Syn-induced neurodegeneration by promoting the dissociation of the calmodulin/Src complex and inhibiting Src, thereby enhancing PP2A activity. This was supported by the observation that PP2A activity was decreased in PD patients, which was negatively correlated with Hoehn and Yahr scores. Our results provide novel insight into the mechanisms underlying neurodegeneration in PD as well as possible avenues for therapeutic intervention in the treatment of this disease.