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Understanding the causes of the decline in bee population has attracted intensive attention worldwide. The indiscriminate use of agrochemicals is a persistent problem due to their physiological and behavioural damage to bees. Glyphosate and its commercial formulation stand out due to their wide use in agricultural areas and non-crop areas, such as parks, railroads, roadsides, industrial sites, and recreational and residential areas, but the mode of action of glyphosate on bees at the molecular level remains largely unelucidated. Here, we found that the numbers of differentially expressed genes and metabolites under glyphosate commercial formulation (GCF) stress were significantly higher in Apis cerana cerana than in Apis mellifera ligustica. Despite these differences, the number of differentially expressed transcripts increased following an increase in the GCF treatment time in both A. cerana cerana and A. mellifera ligustica. GCF exerted adverse impacts on the immune system, digestive system, nervous system, amino acid metabolism, carbohydrate metabolism, growth and development of both bee species by influencing their key genes and metabolites to some extent. The expression of many genes involved in immunity, agrochemical detoxification and resistance, such as antimicrobial peptides, cuticle proteins and cytochrome P450 families, was upregulated by GCF in both bee species. Collectively, our results indicate that both A. cerana cerana and A. mellifera ligustica strive to mitigate the pernicious effects caused by GCF by regulating detoxification and immune systems. Moreover, A. cerana cerana might be better able to withstand the toxic effects of GCF with lower fitness costs than A. mellifera ligustica. Our work will contribute to elucidating the deleterious physiological and behavioural impacts of GCF on bees. Unlabelled Image • Bees were fed with glyphosate commercial formulation (GCF). • GCF had adverse impacts on the digestive system and development of bees. • Bees regulate detoxification and immune system to mitigate pernicious effects. • A. cerana cerana might be better able to withstand GCF than A. mellifera ligustica. [ABSTRACT FROM AUTHOR]