Thiamethoxam, an insecticide with high usage and large amounts of environmental residues, has been reported to affect the pupation and survival of honey bee larvae at sublethal concentrations. The molecular mechanisms are not fully understood. In this study, we measured the response of juvenile hormone (JH) to environmental concentrations of thiamethoxam using liquid chromatography-tandem mass spectrometry (LC-MS/MS), monitored the dynamic changes in the transcription of genes encoding major JH metabolic enzymes (CYP15A1, FAMET, JHAMT and JHE) using RT-qPCR, and analysed the transcriptome changes in worker larvae under thiamethoxam stress using RNA-seq. Thiamethoxam significantly increased the levels of JH3 in honey bee larvae, but no significant changes in the transcript levels of the four major metabolic enzymes were observed. Thiamethoxam exposure resulted in 140 differentially expressed genes (DEGs). P450 CYP6AS5 was upregulated, and some ion-related, odourant-related and gustatory receptors for sugar taste genes were altered significantly. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that amino acid metabolism and protein digestion and absorption were influenced by thiamethoxam. These changes may do harm to honey bee caste differentiation, foraging behaviour related to sensory perception and nutrient levels of bee colonies. These results represent the first assessment of the effects of thiamethoxam on JH in honey bee larvae and provides a new perspective and molecular basis for the study of JH regulation and thiamethoxam toxicity to honey bees.