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Flupyradifurone (FLU) has great application potential in agricultural production as a new generation of neonicotinoid insecticide after imidacloprid. Nevertheless, the toxic effects of FLU on non-target soil organisms remain unclear, resulting in considerable environmental risks. We evaluated the acute and subchronic toxicities of FLU to earthworms. The results of acute toxicity show that the median lethal concentration (LC 50) values (14 d) of FLU were 186.9773 mg kg−1 for adult earthworms and 157.6502 mg kg−1 for juveniles, respectively. The subchronic toxicity of FLU that focused on the activities of antioxidant and detoxication enzymes showed the superoxide dismutase (SOD), catalase (CAT), and glutathione-S transferase (GST) activities in earthworms increased while the peroxidase (POD) and acetylcholinesterase (AChE) activities decreased after exposure to FLU. Oxidative damage analyses revealed that the reactive oxygen species (ROS) level and malonaldehyde (MDA) content in earthworms were increased by FLU, resulting in DNA damage. Transcriptomics and RT-qPCR confirmed that FLU influenced the expression of genes related to antioxidant response and detoxification of earthworms. Ultimately detoxification metabolism, environmental information processing, cell processes, and immune system pathways are significantly enriched to respond jointly to FLU. Our study fills the gaps in the toxicity of FLU to earthworms, providing a basis for its risk assessment of soil ecosystems and non-target biological toxicity. [Display omitted] • Flupyradifurone (FLU) was low toxic to adult and juvenile earthworms from LC 50. • FLU led to oxidative stress, lipid and DNA damage to adult earthworms. • Transcriptomics revealed oxidative stress and detoxification mechanisms in earthworms. • Integrated biomarker response indicated FLU toxicity had dose and time effects. [ABSTRACT FROM AUTHOR]