In this study, we revealed that mesencephalic astrocyte-microglia interaction promoted a pesticide rotenone-induced dopaminergic neurotoxicity. In addition, we demonstrated that mesencephalic astrocytes upregulated alpha-synuclein expression in the dopaminergic neurons. We examined secreted molecules from rotenone-treated mesencephalic astrocyte and microglia co-culture by LC-MS/MS analysis. It is demonstrated increase in the inflammatory molecules and decrease in the antioxidants and a protein which inhibits alpha-synuclein aggregation and cell-to-cell propagation. In rotenone-injected parkinsonian mice, we observed alpha-synuclein aggregation and neurodegeneration not only in the central nervous system but also peripheral enteric nervous system. However, change in the alpha-synuclein expression and neurodegeneration are not parallel in some brain region. These results suggest some factors in addition to alpha-synuclein aggregation are involved in region-specific neurodegeneration.