Therapeutics designed to target α-synuclein (α-syn) aggregation may be critical in halting the progression of pathology in Parkinson's disease (PD) patients. Nanobodies are single-domain antibody fragments that bind with antibody specificity, but allow readier genetic engineering and delivery. When expressed intracellularly as intrabodies, anti-α-syn nanobodies fused to a proteasome-targeting proline, aspartate or glutamate, serine, and threonine (PEST) motif can modulate monomeric concentrations of target proteins. Here we aimed to validate and compare the in vivo therapeutic potential of gene therapy delivery of two proteasome-directed nanobodies selectively targeting α-syn in a synuclein overexpression-based PD model: VH14PEST (non-amyloid component region) and NbSyn87PEST (C-terminal region). Stereotaxic injections of adeno-associated viral 5-α-syn (AAV5-α-syn) into the substantia nigra (SN) were performed in Sprague-Dawley rats that were sorted into three cohorts based on pre-operative behavioral testing. Rats were treated with unilateral SN injections of vectors for VH14PEST, NbSyn87PEST, or injected with saline 3 weeks post lesion. Post-mortem assessments of the SN showed that both nanobodies markedly reduced the level of phosphorylated Serine-129 α-syn labeling relative to saline-treated animals. VH14PEST showed considerable maintenance of striatal dopaminergic tone in comparison to saline-treated and NbSyn87PEST-treated animals as measured by tyrosine hydroxylase immunoreactivity (optical density), DAT immunoreactivity (optical density), and dopamine concentration (high-performance liquid chromatography). Microglial accumulation and inflammatory response, assessed by stereological counts of Iba-1-labeled cells, was modestly increased in NbSyn87PEST-injected rats but not in VH14PEST-treated or saline-treated animals. Modest behavioral rescue was also observed, although there was pronounced variability among individual animals. These data validate in vivo therapeutic efficacy of vector-delivered intracellular nanobodies targeting α-syn misfolding and aggregation in synucleinopathies such as PD.