Rotenone, the inhibitor of mitochondrial complex I, is widely used to develop the experimental models of Parkinson’s disease, but the mechanism of neurotoxicity of rotenone is still not clearly established. In the current study we showed that rotenone mediated mitochondrial dysfunctions and cell death in SH-SY5Y cells (human neuroblastoma cell line) over an incubation period of 48 h are triggered by the accumulation of α-synuclein, and silencing of α-synuclein protein expression by specific siRNA abolished the cytotoxic effects of rotenone. Based on the current and earlier published data, the deleterious action of α-synuclein in SH-SY5Y cells has been ascribed to the activation of mitochondrial permeability transition pore (mPTP). In rotenone induced rat model of Parkinson’s disease, nigral dopaminergic neuronal death and altered functions of isolated midbrain mitochondria such as mitochondrial membrane depolarization, decreased ATP synthesis, increased ROS production and inhibition of complex I-III were observed which could be prevented markedly (except complex I-III inhibition) by cyclosporine A which is a well-known blocker of mPTP. The nigral accumulation of α-synuclein after rotenone treatment of rats, however, was not noticeably prevented by cyclosporine A. Additionally, cyclosporine A prevented neuroinflammation in the midbrain region of rotenone treated rats. These results have implications in the pathogenesis and also highlight the therapeutic potential of cyclosporine A for Parkinson’s disease.