Zinc oxide nanoparticles (ZnO NPs) are increasingly applied in a diverse array of industrial and commercial products. Therefore, it is urgently required to characterize their toxic behavior. ZnO NPs have been reported to induce toxic effects at the levels of the individual organism, tissue, cell and DNA. However, little is known about the potential impacts of ZnO NPs at a subcellular level. In the present work, we investigated the toxicity of ZnO NPs to the isolated rat liver mitochondria. We found that treatment of mitochondria with ZnO NPs resulted in collapse of mitochondrial membrane potential (Δψ), swelling, depression of respiration, inner membrane permeabilization to H+ and K+, alterations of ultrastructure, release of cytochrome c, generation of reactive oxygen species (ROS), and Zn2+ liberation from ZnO NPs. These results suggested that ZnO NPs can increase the inner membrane permeability and impair the respiratory chain, thus leading to energy dissipation, oxidative stress and even apoptosis. This putative mechanism helps us learn more about the toxicology of this nanomaterial.