Parkin is known to mitigate alpha-synuclein-induced neuronal cell death in vitro, which suggests that the parkin gene therapy is a candidate for therapeutic strategies for Parkinson's disease (PD). In the present study, the parkin gene therapy was investigated for its ameliorative effects on alpha-synucleinopathy in substantia nigra (SN) of rats. A recombinant adeno-associated viral (rAAV) vector system has frequently been used for the gene transfer to rat SN, and we have previously demonstrated that this technique induced the alpha-synucleinopathy, which closely resembles pathogenetic changes in PD. Therefore, in the present study, the effect of parkin was examined by co-infection of rAAV-parkin with rAAV-alpha-synuclein into dopaminergic neurons in SN. At 13 weeks post-rAAV infection, alpha-synuclein overexpression induced dopaminergic neuron loss, while co-expression of parkin mitigated the alpha-synuclein toxicity. Moreover, alpha-synuclein-induced dopaminergic neuron loss consequently resulted in motor dysfunction, which was also mitigated by parkin. Taken together, our results indicate that the parkin gene therapy is effective against alpha-synucleinopathy, suggesting its potential suitability for patients with PD.