Neonicotinoid toxicity to honeybees varies among active ingredients, with the insects being tolerant to thiacloprid but sensitive to imidacloprid. The selective toxicity has been hypothesized to be due to differences in metabolic detoxification by cytochrome P450s. In vitro experiments revealed that CYP9Q3 efficiently metabolises thiacloprid but not imidacloprid, consistent with the toxicity data. Honeybee tolerance to tau-fluvalinate, a pyrethroid acaricide used against Varroa destructor, has also been linked to P450-mediated detoxification. To address the role of CYP9Q3 in vivo in Apis mellifera, we used an RNA interference approach based on double-stranded RNA delivery through the median ocellus of adults to transiently suppress expression of CYP9Q3 in the head. Following confirmation of CYP9Q3 suppression by qPCR, fixed-dose topical bioassays were conducted with the neonicotinoids imidacloprid and thiacloprid and the pyrethroid tau-fluvalinate. Suppression of CYP9Q3 was associated with increased mortality following exposure to thiacloprid and tau-fluvalinate, whereas no increase was observed with imidacloprid. These findings provide in vivo functional evidence supporting the role of CYP9Q3 in honeybee tolerance to thiacloprid and tau-fluvalinate. The RNAi approach offers a valuable tool for investigating the functions of cytochrome P450 enzymes and other genes expressed in the honey bee head that are involved in insecticide susceptibility.