Abstract: Neonicotinoid insecticides are now the most widely used insecticides in the world. Previous studies have indicated that sublethal doses of neonicotinoids impair learning, memory capacity, foraging and immunocompetence in honeybees (Apis mellifera). Despite this, few studies have been carried out on the molecular effects of neonicotinoids. In this study, we focus on the second-generation neonicotinoid thiamethoxam, which is currently widely used in agriculture to protect crops. Using high-throughput RNA-Seq, we investigated the transcriptome profile of honeybees after subchronic exposure to thiamethoxam (10 ppb) over 10 days. In total, 609 differentially-expressed genes (DEGs) were identified, of which 225 were up-regulated and 384 were down-regulated. The functions of some DEGs were identified, and GO enrichment analysis showed that the enriched DEGs were mainly linked to metabolism, biosynthesis and translation. KEGG pathway analysis showed that thiamethoxam affected biological processes including ribosomes, the oxidative phosphorylation pathway, tyrosine metabolism pathway, pentose and glucuronate interconversions and drug metabolism. Overall, our results provide a basis for understanding the molecular mechanisms of the complex interactions between neonicotinoid insecticides and honeybees.