Several studies reported that pesticide exposure, such as rotenone or paraquat, increased the risk of Parkinson's disease (PD). Rotenone is used to develop animal models of PD because the pesticide induces PD-like pathology. It is well-known that the inhibition of mitochondrial complex I is involved in rotenone-induced dopaminergic neurotoxicity. However, the mechanism underlying selective dopaminergic neurotoxicity by rotenone exposure remains unknown. In this study, we examined involvement of glial cells in rotenone-induced dopaminergic neurotoxicity using primary cultured cells. We prepared neuronal culture, neuron-glia co-culture, glial cell culture (astrocyte+microglia), astrocyte culture, and microglia culture from mesencephalon of SD rats embryos at 15 days of gestation. Rotenone exposure significantly decreased dopaminergic neurons in neuron-glia co-cultures, but not in enriched neuronal cultures. Dopaminergic neurotoxicity was also induced by treatment with conditioned media from rotenone-treated glial cells. In contrast, conditioned media from rotenone-treated astrocytes or microglia had no effect on dopaminergic neurons survival. These results suggest that astrocyte-microglia interaction could play an important role in rotenone-induced dopaminergic neurodegeneration.