Effects of silencing ectopically expressed hSNCA in rat substantia nigra (SN) were examined as a novel therapeutic approach to Parkinson's disease (PD). AAV-hSNCA with or without an AAV harboring a short-hairpin (sh)RNA targeting hSNCA or luciferase was injected into one SN. At 9 weeks, hSNCA-expressing rats had reduced SN dopamine (DA) neurons and exhibited a forelimb deficit. AAV-shRNA-SNCA silenced hSNCA and protected against the forelimb deficit. However, AAV-shRNA-SNCA also led to DA neuron loss suggesting undesirable effects of chronic shRNA expression. Effects on nigrostriatal-projecting neurons were examined using a retrograde tract tracer. Loss of striatal-projecting DA neurons was evident in the vector injection site, whereas DA neurons outside this site were lost in hSNCA-expressing rats, but not in hSNCA-silenced rats. These observations suggest that high levels of shRNA-SNCA were toxic to DA neurons, while neighboring neurons exposed to lower levels were protected by hSNCA gene silencing. Also, data collected on DA levels suggest that neurons other than or in addition to nigrostriatal DA neurons contributed to protection of forelimb use. Our observations suggest that while hSNCA gene silencing in DA neurons holds promise as a novel PD therapy, further development of silencing technology is required.