AbstractGlobally, neonicotinoids are the most used insecticides, despite their well-documented sub-lethal effects on beneficial insects. Neonicotinoids are nicotinic acetylcholine receptor agonists. Memory, circadian rhythmicity and sleep are essential for efficient foraging and pollination and require nicotinic acetylcholine receptor signalling. The effect of field-relevant concentrations of the European Union-banned neonicotinoids: imidacloprid, clothianidin, thiamethoxam and thiacloprid were tested on Drosophila memory, circadian rhythms and sleep. Field-relevant concentrations of imidacloprid, clothianidin and thiamethoxam disrupted learning, behavioural rhythmicity and sleep whilst thiacloprid exposure only affected sleep. Exposure to imidacloprid and clothianidin prevented the day/night remodelling and accumulation of pigment dispersing factor (PDF) neuropeptide in the dorsal terminals of clock neurons. Knockdown of the neonicotinoid susceptible Dα1 and Dβ2 nicotinic acetylcholine receptor subunits in the mushroom bodies or clock neurons recapitulated the neonicotinoid like deficits in memory or sleep/circadian behaviour respectively. Disruption of learning, circadian rhythmicity and sleep are likely to have far-reaching detrimental effects on beneficial insects in the field.