Abstract The silkworm Bombyx mori L. is a model organism of the order Lepidoptera. Understanding the mechanism of pesticide resistance in silkworms is valuable for Lepidopteran pest control. In this study, comparative metabolomics was used to analyze the metabolites of 2 silkworm strains with different pesticide resistance levels at 6, 12, and 24 h after feeding with fenpropathrin. Twenty‐six of 27 metabolites showed significant differences after fenpropathrin treatment and were classified into 6 metabolic pathways: glycerophospholipid metabolism, sulfur metabolism, glycolysis, amino acid metabolism, the urea cycle, and the tricarboxylic acid (TCA) cycle. After analyzing the percentage changes in the metabolic pathways at the 3 time points, sulfur metabolism, glycolysis, and the TCA cycle showed significant responses to fenpropathrin. Confirmatory experiments were performed by feeding silkworms with key metabolites of the 3 pathways. The combination of iron(II) fumarate + folic acid (IF‐FA) enhanced fenpropathrin resistance in silkworms 6.38 fold, indicating that the TCA cycle is the core pathway associated with resistance. Furthermore, the disruption of several energy‐related metabolic pathways caused by fenpropathrin was shown to be recovered by IF‐FA in vitro. Therefore, IF‐FA may have a role in boosting silkworm pesticide resistance by modulating the equilibrium between the TCA cycle and its related metabolic pathways.
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