Declines in honey bee populations represent a worldwide concern. The widespread use of neonicotinoid insecticides has been one of the factors linked to these declines. Sublethal doses of a neonicotinoid insecticide, imidacloprid, has been reported to cause olfactory learning deficits in honey bees via impairment of the target organ, the brain. In the present study, olfactory learning of honey bees was compared between controls and imidacloprid-treated bees. The brains of imidacloprid-treated and control bees were used for comparative transcriptome analysis by RNA-Seq to elucidate the effects of imidacloprid on honey bee learning capacity. The results showed that the learning performance of imidacloprid-treated bees was significantly impaired in comparison with control bees after chronic oral exposure to imidacloprid (0.02 ng/μl) for 11 days. Gene expression profiles between imidacloprid treatment and the control revealed that 131 genes were differentially expressed, of which 130 were downregulated in imidacloprid-treated bees. Validation of the RNA-Seq data using qRT-PCR showed that the results of qRT-PCR and RNA-Seq exhibited a high level of agreement. Gene ontology annotation indicated that the oxidation-reduction imbalance might exist in the brain of honey bees due to oxidative stress induced by imidacloprid exposure. KEGG and ingenuity pathway analysis revealed that transient receptor potential and Arrestin 2 in the phototransduction pathway were significantly downregulated in imidacloprid-treated bees, and that five downregulated genes have causal effects on behavioral response inhibition in imidacloprid-treated bees. Our results suggest that downregulation of brain genes involved in immune, detoxification and chemosensory responses may result in decreased olfactory learning capabilities in imidacloprid-treated bees.