BACKGROUNDThe insecticides spinosad and imidacloprid are neurotoxins with distinct modes of action. Both target nicotinic acetylcholine receptors (nAChRs), albeit different subunits. Spinosad is an allosteric modulator, that upon binding initiates endocytosis of its target, nAChRα6. Imidacloprid binding triggers excessive neuronal ion influx. Despite these differences, low‐dose effects converge downstream in the precipitation of oxidative stress and neurodegeneration.RESULTSUsing RNA‐sequencing, we compared the transcriptional signatures of spinosad and imidacloprid, at low‐dose exposures. Both insecticides cause up‐regulation of glutathione S‐transferase and cytochrome P450 genes in the brain and down‐regulation in the fat body, whereas reduced expression of immune‐related genes is observed in both tissues. Spinosad shows unique impacts on genes involved in lysosomal function, protein folding, and reproduction. Co‐expression analyses revealed little to no correlation between genes affected by spinosad and nAChRα6 expressing neurons, but a positive correlation with glial cell markers. We also detected and experimentally confirmed nAChRα6 expression in fat body cells and male germline cells. This led us to uncover lysosomal dysfunction in the fat body following spinosad exposure, and a fitness cost in spinosad‐resistant (nAChRα6 null) males – oxidative stress in testes, and reduced fertility.CONCLUSIONSpinosad and imidacloprid share transcriptional perturbations in immunity‐, energy homeostasis‐, and oxidative stress‐related genes. Low doses of other neurotoxic insecticides should be investigated for similar impacts. While target‐site spinosad resistance mutation has evolved in the field, this may have a fitness cost. Our findings demonstrate the power of tissue‐specific transcriptomics approach and the use of single‐cell transcriptome data. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.