Most of &alpha-synuclein deposited as Lewy bodies, a hallmark of Parkinson's disease (PD), is phosphorylated at Ser129. Ser129-phosphorylation is a sensitive and specific marker for detecting abnormal aggregation of &alpha-synuclein in human tissues and experimental models of PD. However, it remains unclear whether or not this modification has a role in the biogenesis of Lewy bodies and neurodegeneration of PD. To find a clue for this issue, we assessed the impact of Ser129-phosphorylation on the functions of &alpha-synuclein. First, we investigated the effect of this Ser129-phosphorylation on dopamine uptake in dopaminergic SH-SY5Y cells. Our data showed that membrane-associated &alpha-synuclein enhanced dopamine uptake capacity of dopamine transporter by G-protein-coupled receptor kinases (GRKs)-mediated Ser129-phosphorylation, whereas the phosphorylation by casein kinase 2 had no effect. This suggests that &alpha-synuclein modulates intracellular dopamine levels with no functional redundancy in Ser129-phosphorylation between these kinases. Then, we investigated the metabolic fate of Ser129-phosphorylated &alpha-synuclein in SH-SY5Y cells. Experiments using cycloheximide showed that Ser129-phosphorylated &alpha-synuclein diminished rapidly, in contrast to the stable expression of total &alpha-synuclein. The short half-life of Ser129-phosphorylated &alpha-synuclein was blocked by proteasome inhibitor, MG132, to a greater extent than protein phosphatase 2A/1 inhibitor, okadaic acid. In rat primary cortical neurons, either MG132, lactacystin, or okadaic acid accumulated Ser129-phosphorylated α-synuclein. These data showed that Ser129-phosphorylated &alpha-synuclein is targeted to the proteasome pathway, in addition to undergoing dephosphorylation. Finally, we assessed the effect of Ser129-phosphorylation on &alpha-synuclein neurotoxicity in an AAV-based rat model. We found that GRK6-mediated authentic Ser129-phosphorylation significantly enhanced A53T mutant &alpha-synuclein-induced degeneration of dopaminergic neurons. Taken together, our data suggest that Ser129-phosphorylation is not only the useful marker for abnormal accumulation of &alpha-synuclein but also a modulator of physiological and pathological functions of the molecule.