As a new threat to environment all through the world, perchlorate (ClO4(-)) was predominantly a thyrotoxin, and its toxic manifestations in non-thyroid were also documented. To date, little is known about the effect of ClO4(-) on cell and organelle. To reveal the toxicity of ClO4(-) on living organism in-depth, mitochondria isolated from liver of Carassius auratus were incubated with different concentrations of ClO4(-). The results demonstrated that ClO4(-)-induced mitochondrial oxidative stress, and subsequently caused a gradual opening of permeability transition pore leading to mitochondrial swelling and lipid peroxidative membrane damage. ClO4(-) has a conspicuous inhibition of electron transport chain activity which largely correlated to complexes I and IV. The investigations clearly demonstrated the oxidative stress of ClO4(-) in mitochondria, may well reveal cytotoxic effects in vitro that merit further investigation.