Rotenone, a complex I inhibitor, induces Parkinson's disease (PD)-like features in rats, including dopaminergic neuron loss and α-synuclein-positive inclusions. This study investigates rotenone toxicity using three models: SK-N-MC neuroblastoma cells, organotypic midbrain slice cultures, and rotenone-treated animals. In neuroblastoma cells, rotenone caused dose-dependent ATP depletion, oxidative damage, and cell death. However, rotenone-insensitive NDI1, a yeast NADH dehydrogenase, protected cells from toxicity, indicating rotenone's action at complex I. Oxidative damage, not ATP depletion, was responsible for cell death, as antioxidants and NDI1 transfection blocked toxicity. In midbrain slice cultures, rotenone caused oxidative damage and dopaminergic neuron loss, which were prevented by α-tocopherol. In rotenone-treated animals, oxidative damage was observed in the midbrain and olfactory bulb, regions affected in PD. These findings suggest that oxidative damage, mediated by complex I dysfunction, is central to rotenone-induced neurotoxicity. Antioxidants like α-tocopherol and coenzyme Q10 protected against rotenone toxicity, highlighting their potential as therapies for PD. The study supports the role of oxidative stress in PD pathogenesis and underscores the importance of targeting oxidative damage in PD treatment.Rotenone, a complex I inhibitor, induces Parkinson's disease (PD)-like features in rats, including dopaminergic neuron loss and α-synuclein-positive inclusions. This study investigates rotenone toxicity using three models: SK-N-MC neuroblastoma cells, organotypic midbrain slice cultures, and rotenone-treated animals. In neuroblastoma cells, rotenone caused dose-dependent ATP depletion, oxidative damage, and cell death. However, rotenone-insensitive NDI1, a yeast NADH dehydrogenase, protected cells from toxicity, indicating rotenone's action at complex I. Oxidative damage, not ATP depletion, was responsible for cell death, as antioxidants and NDI1 transfection blocked toxicity. In midbrain slice cultures, rotenone caused oxidative damage and dopaminergic neuron loss, which were prevented by α-tocopherol. In rotenone-treated animals, oxidative damage was observed in the midbrain and olfactory bulb, regions affected in PD. These findings suggest that oxidative damage, mediated by complex I dysfunction, is central to rotenone-induced neurotoxicity. Antioxidants like α-tocopherol and coenzyme Q10 protected against rotenone toxicity, highlighting their potential as therapies for PD. The study supports the role of oxidative stress in PD pathogenesis and underscores the importance of targeting oxidative damage in PD treatment.