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