The decreasing number of bees is a global ecological problem. With the advancement of agricultural modernisation, the large-scale use of neonicotinoid insecticides is one of the main factors leading to the decline of bees. The aim of the present study was to investigate the effect and the mechanisms underlying bees impaired by dinotefuran. Acute (48 h) oral toxicity tests showed that a 5% lethal concentration (LC₅) was 0.220 mg/L, and a 20% lethal concentration (LC₂₀) was 0.458 mg/L. The gene expression profile shows that when compared with the control group, the LC₅ group induced 206 significantly upregulated, differentially expressed genes (DEGs) and 363 significantly downregulated DEGs, while the LC₂₀ group induced 180 significantly upregulated DEGs and 419 significantly downregulated DEGs. Significantly, transcriptomic analysis revealed DEGs involved in immunity, detoxification, and the nervous system, such as antimicrobial peptides, vitellogenin, synaptotagmin-10, AChE-2, and nAChRa9. Furthermore, Gene Ontology (GO) annotation and Kyoto Encyclopaedia of Genes and Genomes (KEGG) pathway analysis revealed that DEGs were enriched in amino acid and fatty acid biosynthesis and metabolism pathways. Collectively, our findings will help clarify the deleterious physiological and behavioural impacts of dinotefuran on bees and provide a basis for future research on the mechanisms underlying bees impaired by dinotefuran.