BACKGROUND: Beneficial insects, including pollinators, encounter various pesticide exposure conditions, from brief high-concentration acute exposure to continuous low-level chronic exposure. To effectively assess the environmental risks of pesticides, it is critical to understand how different exposure schemes influence their effects. Unfortunately, this knowledge remains limited. To clarify whether different exposure schemes disrupt the physiology of pollinators in a similar manner, we exposed bumble bees to acute or chronic treatments of three different pesticides: acetamiprid, clothianidin, or sulfoxaflor. Genome-wide gene expression profiling enabled us to compare the effects of these treatments on the brain in a high-resolution manner. RESULTS: There were two main findings: First, acute and chronic exposure schemes largely affected non-overlapping sets of genes. Second, different pesticides under the same exposure scheme showed more comparable effects than the same pesticide under different exposure schemes. Each exposure scheme induced a distinct gene expression profile. Acute exposure mainly caused upregulation of genes linked to the stress response mechanisms, like peroxidase and detoxification genes, while chronic exposure predominantly affected immunity and energy metabolism. CONCLUSIONS: Our findings show that the mode of exposure is critical in determining the molecular effects of pesticides. These results signal the need for safety testing practices to better consider mode-of-exposure dependent effects and suggest that transcriptomics can support such improvements.