Sublethal effect considered as an emerging factor to assess the environmental risk of insecticides, which can impact the insects on both physiology and behavior. Lethal exposure can be causing near immediate mortality. Pests are inevitably exposed to sublethal and lethal dose in the agroecosystem following application of pesticides. Insecticides, widely used for the control of insect pests, are irreplaceable in insect pest management. The effects of imidacloprid by the method of high-throughput non-targeted metabolomics was investigated in Aphis gossypii Glover exposed to LC10 and LC90 doses of the imidacloprid, and the control group was treated with the same condition without imidacloprid. Pairwise comparisons showed that 111 metabolites changed significantly, 60 in the LC10 group, and 66 in the LC90 group compared to the control group, while only 16 changes in the LC10 were same with that in LC90 group. Among the changed metabolites, a total of 16 metabolites were identified as potential biomarkers, which represented the most influential pathways including glycolysis and gluconeogenesis, alanine, aspartate, and glutamate metabolism, ascorbate and aldarate metabolism, glutathione metabolism, phenylalanine metabolism, tyrosine metabolism, caffeine metabolism and parkinson’s disease (PD), which could account for the sublethal and lethal effects on A. gossypii. These modified metabolic pathways demonstrated that high energy consumption, excitotoxicity and oxidative stress (OS) were appeared in both LC10 and LC90 groups, while PD was detected only in the LC90 group. The results of non-targeted metabolomics revealed the effects of neonicotinoid pesticide exposure on A. gossypii successfully, and provided a deep insight into the influenced physiology by the stress of neonicotinoid pesticide in the insect.