Recently, environmental risk and toxicity of neonicotinoid insecticides to honey bees have attracted extensive attention. However, toxicological understanding of neonicotinoid insecticides on gut microbiota is limited. In the present study, honey bees (Apis mellifera L.) were exposed to a series of nitenpyram for 14 days. Results indicated that nitenpyram exposure decreased the survival and food consumption of honey bees. Furthermore, 16S rRNA gene sequencing revealed that nitenpyram caused significant alterations in the relative abundance of several key gut microbiotas, which contribute to metabolic homeostasis and immunity. Using high-throughput RNA-Seq transcriptomic analysis, we identified a total of 526 differentially expressed genes (DEGs) that were significantly altered between nitenpyram-treated and control honey bee gut, including several genes related to metabolic, detoxification and immunity. In addition, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed nitenpyram affected several biological processes, of which most were related to metabolism. Collectively, our study demonstrates that the dysbiosis of gut microbiota in honey bee caused by nitenpyram may influence metabolic homeostasis and immunity of bees, and further decrease food consumption and survival of bees.