AbstractWith the rising occurrence of sudden heat waves, honey bees are at risk of exposure to unprecedented heat stress. We investigated the synergistic effects of imidacloprid (IMD) and high temperature on honey bees. Mini hives were treated with IMD (20 ppb for 14 days) and high temperature (41 °C for 6 h) either singly or in combination. Heat shock protein 70 and 90 genes were upregulated in bees exposed to the combined treatment compared to those exposed to each single treatment. Transcriptome analysis revealed that metabolic pathways remained intact in the high temperature treatment, whereas several metabolic pathways were altered by either the IMD treatment (downregulation of cellular respiration pathways) or the combined treatment (upregulation of protein synthesis and signaling pathways). These findings suggest that IMD and high temperature have negative synergistic effects on honey bees.