The hyperphosphorylation of α-synuclein (α-Syn) at serine 129 is associated with in its aggregation, culminating in the formation of Lewy bodies (LBs) in Parkinson's disease (PD) and dementia with LBs. Plasma homocysteine (Hcy) levels are typically high in PD, particularly in those treated with Levodopa. Therefore, we aimed to investigate the effects of Hcy on phosphorylation and aggregation of α-Syn. The human neuroblastoma cell line 3D5 expressing wild-type α-Syn under the control of a tetracycline-off system was treated with Hcy, and aggregation and phosphorylation of α-Syn were examined. Hcy was cytotoxic to 3D5 cells, and Hcy induced cell shrinkage at a concentration >100 ‍μM. Treatment with Hcy upregulated the levels of α-Syn, increased its phosphorylation at serine 129, and increased casein kinase 2A. Moreover, Hcy treatment significantly increased the level of aggregated form of α-Syn, including oligomers in 3D5 cells. However, folate significantly reversed the Hcy-mediated increase in the levels of total α-Syn and its phosphorylation. In conclusion, Hcy enhances the total α-Syn level, and phosphorylated and oligomeric α-Syn formation, thereby promoting LBs formation.