Parkinson's disease (PD) is a devastating neurodegenerative disorder primarily characterized by the progressive and unstoppable loss of dopaminergic (DA) neurons in the substantia nigra. We previously identified NATO3 (FERD3L), a conserved developmental transcription factor, as essential for maintaining DA neuron function during aging. Here, we show that AAV-mediated Nato3 gene transfer into the mouse substantia nigra prevents DA neuron degeneration in both MPTP-induced and α-synuclein (α-Syn) overexpression PD models. This neuroprotective effect is achieved by improving autophagic flux and α-Syn clearance. Furthermore, lentiviral-mediated NATO3 overexpression in human midbrain DA neurons, derived from induced pluripotent stem cells carrying the pathological α-Syn A53T mutation, effectively reversed key disease hallmarks. These include α-Syn accumulation, aberrant mitochondrial morphology, autophagic impairments, and compromised neurite structure. Collectively, these in vivo and in vitro findings highlight NATO3's role in safeguarding DA neurons against pathological cellular events, positioning NATO3 as a therapeutic target for PD.