Oxydemeton-methyl, is an organothiophosphate insecticide, which is widely used in agricultural and urban pest controls. It exists in the environment and a large amount bioaccumulation in the wildlife due to its strong water solubility and mobility. Although its potentially harmful effect on animals and humans, few studies have focused on the oxydemeton-methyl pollution in the environment. Zebrafish have been used for many years to valuate the pollution status of water and toxicity of chemicals. In the present study, we aimed to investigate the effect of oxydemeton-methyl on the expression level of liver microsomal cytochrome P450, on the activity of NADPH-P450 reductase and reactive oxygen species (ROS) generation in zebrafish. Adult male and female zebrafish were treated with different concentration of oxydemeton-methyl (10, 50, 100 μM) for 5, 10, 20 and 30 days. We found that the oxydemeton-methyl exposure significantly increased the P450 levels and the activity of NAPDH-P450 reductase. ROS generation and the DNA damage were augmented in a dose-dependent manner in the zebrafish. These results indicated that oxydemeton-methyl is able to induce strong oxidative stress and hence highly toxic to the zebrafish.