Methylmercury induces oxidative stress and subsequent neuronal injury. However, the mechanism by which methylmercury elicits reactive oxygen species (ROS) production remains under debate. In this study, we investigated the involvement of mitochondrial ROS in methylmercury-induced neuronal cell injury using human neuroblastoma SH-SY5Y-derived ρ⁰ cells, which have a deletion of mitochondrial DNA and thus decreased respiratory activity. SH-SY5Y cells were cultured for 60 days in the presence of ethidium bromide to produce ρ⁰ cells. Our ρ⁰ cells showed decreases in the cytochrome c oxidase expression and activity as well as oxygen consumption compared with original SH-SY5Y cells. Methylmercury at a concentration of 1 µM induced cell death with oxidative stress in original SH-SY5Y cells, but not ρ⁰ cells, indicating that ρ⁰ cells are resistant to methylmercury-induced oxidative stress. ρ⁰ cells also showed tolerance against hydrogen peroxide and superoxide anion, suggesting that ρ⁰ cells are resistant to total ROS. These data indicate that mitochondrial ROS are clearly involved in oxidative stress and subsequent cell death induced by methylmercury. Considering that the dominant mechanism of ROS generation elicited by methylmercury is due to direct antioxidant enzyme inhibition, mitochondria might play a role in amplifying ROS in methylmercury-induced neurotoxicity.