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The anthranilic diamide insecticide chlorantraniliprole is highly effective against Lepidoptera pests, but the underlying mechanisms of toxic effects of chlorantraniliprole exposures for adapting to the chemical environment are poorly known in fall armyworm (FAW), Spodoptera frugiperda (J.E.Smith). FAW being one of the most pests of maize in Latin America, suddenly appeared in China in 2019 and spread rapidly. In this study, using bioassay and transcriptomic and biochemical analyses, we comprehensively investigated gene expression changes of third instar larvae in response to different sublethal concentrations (LC10 and LC30) of chlorantraniliprole in this insect. Exposure to LC10 chlorantraniliprole (0.73 mg/L) causes 1266 differentially expressed genes (DEGs), of which 578 are up-regulated and 688 down-regulated. Exposure to LC30 (2.49 mg/L) causes differential expression of 3637 DEGs (1545 up-, 2092 down-regulated). Interestingly, the LC30 treatment led to a significant increase in the number of DEGs compared to that of the LC10, indicating a concentration effect manner. Moreover, enrichment analysis identified important DEGs belonging to specific categories, such as amino acid, carbohydrate, lipid, energy, xenobiotics metabolisms, signal transduction, and posttranslational modification pathways, and enzymes activities in enriched pathways were significantly altered at the LC10 and LC30, which matched transcriptome analysis to mediate toxic mechanisms. The DEGs encoding detoxification-related genes were identified and validated by quantitative real-time PCR (qRT-PCR), which correlated with the RNA-sequencing (RNA-seq) data. To our knowledge, these findings provide the first toxicity mechanisms for a better understanding of chlorantraniliprole action and detoxification in FAW and other insect pests at molecular level.