Mesotrione, a triketone herbicide, has been widely used in corn fields in recent years. The present study evaluated the toxicity of mesotrione to earthworms after exposing for 28 d at 0, 0.1, 1.0 and 2.5 mg/kg in a soil mixture (10% sphagnum peat, 20% kaolinite clay, and 70% sand by weight). The levels of reactive oxygen species (ROS), detoxifying enzyme glutathione S-transferase (GST), antioxidant enzyme [superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD)], lipid peroxidation and DNA damage in earthworms were determined. The ROS levels in mesotrione-exposed groups were higher than the control during the whole experiment, however, the SOD activities exhibited a downward trend. The MDA content, GST, CAT and POD activities were elevated at 7th day, but returned to control level after 28 d. The damage of DNA was increased greatly with increasing doses and time, which showed the relationship between dose-effect and time-effect. The results suggested herbicide mesotrione can cause ecological damage to soils as attested by oxidative stress and DNA damage in earthworms.