Increased expression of α-synuclein and point mutations in its amino acid sequence play a causative role in familial forms of Parkinson's disease (PD). In addition, circumstances affecting the level of α-synuclein expression significantly increase the risk of developing sporadic PD. This is further corroborated by the abundance of α-synuclein in protein aggregates called Lewy bodies (LBs), a histological hallmark of the disease. Aging constitutes the most prominent risk factor in the pathogenesis of PD and other synucleinopathies. Protective stress response and repair pathways gradually decline over time, a process likely to contribute to the appearance of neurodegenerative disease. Studies in lower organisms, such as the yeast and the worm Caenorhabditis elegans, have highlighted the role of the evolutionarily conserved transcription factor FoxO3a in the aging process, increasing life span and reducing molecular processes that possibly underlie neurodegeneration in higher organisms. This transcription factor is mainly controlled by insulin/Insulin-like growth factor (IGF) signaling (IIS) and impacts a broad range of disease-related responses involving neuronal apoptosis and adaptation to stress. We have hypothesized that FoxO3a activity may regulate the response of dopaminergic neurons to PD-related stress such as reactive oxygen species (ROS) and α-synuclein proteotoxicity. To address this question, AAV2/6 vectors encoding various forms of FoxO3a were used to modulate the activity of this transcription factor in the adult rat substantia nigra pars compacta (SNpc). We determined a viral dose where expression of the wild-type form of FoxO3a did not impair the survival of nigral neurons and their striatal projections. In contrast, a constitutively active FoxO3a mutant (FoxOm), with an Akt-independent nuclear import, led to a significant loss of dopaminergic cell bodies, consistent with the pro-apoptotic activity of FoxO3a. Expression of a dominant-negative competitor composed of the FoxO DNA-binding domain (FoxODBD) did not significantly impair the survival of nigral neurons. However, at a high viral dose, FoxO inhibition led to neuronal accumulation of 4-hydroxynonenal (HNE), an indicator of chronic oxidative stress, and to a loss of neurons positive for dopaminergic markers. Altogether, these results confirm in nigral neurons the critical role of FoxO3a activity at the intersection between apoptotic response and neuronal resistance to oxidative stress. Next, we hypothesized that FoxO3a may regulate the response of dopaminergic neurons to α-synuclein proteotoxicity. To investigate the effect of FoxO3a on α-synuclein accumulation, we co-injected AAV2/6 vectors modulating FoxO3a activity with a wild-type human α-synuclein vector. When compared to control and FoxOm, both wild-type FoxO3a and FoxODBD significantly reduced the α-synuclein-induced loss of neurons positive for dopaminergic markers. Importantly, the injection of vectors activating FoxO3a (wild-type and FoxOm) led to an overall reduction in soluble α-synuclein monomeric forms, concomitant with the formation of dense proteinase K-resistant aggregates found in the neuronal cell soma. With the dominant negative FoxODBD, we observed an accumulation of α-synuclein, with a diffuse distribution of proteinase K-resistant immunoreactivity in both axon and neuronal soma. These data point to FoxO3a as an important regulator of neuronal survival in the SNpc, which can oppose α-synuclein accumulation and proteotoxicity. The role of FoxO3a in α-synuclein toxicity may provide some clues about the role of the aging process in PD neurodegeneration.