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As a common defense mechanism in Hymenoptera, bee venom has complex components. Systematic and comprehensive analysis of bee venom components can aid in early evaluation, accurate diagnosis, and protection of organ function in humans in cases of bee stings. To determine the differences in bee venom composition and metabolic pathways between Apis cerana and Apis mellifera, proton nuclear magnetic resonance (¹H‐NMR) technology was used to detect the metabolites in venom samples. A total of 74 metabolites were identified and structurally analyzed in the venom of A. cerana and A. mellifera. Differences in the composition and abundance of major components of bee venom from A. cerana and A. mellifera were mapped to four main metabolic pathways: valine, leucine and isoleucine biosynthesis; glycine, serine and threonine metabolism; alanine, aspartate and glutamate metabolism; and the …