Cyantraniliprole is a second-generation diamide insecticide that exhibited excellent biological efficacy against a variety of pests. To assess the toxic impact of cyantraniliprole on earthworms, the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), activities of superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST), as well as DNA damage were measured after exposed to five cyantraniliprole concentrations ranging from 0 to 10.00 mg/kg for 7, 14, 21 and 28 days. In most treatment groups, the ROS levels increased significantly before exposure time of 14 days and then returned to normal levels. However, the SOD and CAT activities showed different response with activities were first significantly decreased and subsequently increased. The peroxidase (POD) activity showed no significant differences between treatment and control groups at first and then significantly increased. However, the opposite pattern characterized the GST activity. Also, maybe being dose-dependent before 14 days. The MDA concentration was used as a measure of lipid peroxidation (LPO). During experiment period, the MDA concentrations significantly increased when treated by this pesticide. The olive tail moment (OTM) was used as a measure of DNA damage. At higher concentrations of cyantraniliprole and longer exposure times, the OTM gradually increased, and DNA damage in the earthworms gradually increased. The weight of the high-dose (i.e., 5 mg/kg, 10 mg/kg) earthworms showed a significant trend of decrease phenomenon. Overall, the results suggest that sub-chronic exposure to cyantraniliprole causes DNA damage and LPO, weight loss and growth inhibition, leading to antioxidant defence responses in earthworms.