[Aim]  To elucidate the molecular mechanisms underlying diet-induced physiological regulation in a royal jelly-producing strain of honeybee (Apis mellifera ligustica), and thereby improve the husbandry of this strain. [Methods]  The effects of three different diets on the hemolymph metabolome and proteome of the royal jelly-producing strain were investigated. Caged newly emerged worker bees were randomly assigned to one of four dietary treatments: Camellia bee pollen, rapeseed bee pollen, soybean meal, or a protein-free, control diet. Hemolymph samples were collected from 9-day-old bees and analyzed using ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS)-based, untargeted metabolomics and proteomics, to systematically compare metabolite and protein profiles among the treatment groups. [Results]  A total of 129 metabolites were identified. Compared to the control group, all treatment groups had significantly higher phenylalanine, tyrosine and tryptophan biosynthesis, activity (P < 0.05). Notably, the α‑linolenic acid metabolism was markedly upregulated in the rapeseed bee pollen-fed group relative to the soybean meal-fed group (P < 0.05). A total of 640 proteins were identified and quantified by proteomics analysis. All dietary groups showed common enrichment in core metabolic pathways, including carbon metabolism, amino acid biosynthesis and glycolysis/gluconeogenesis. Furthermore, the bee pollen‑fed groups had stronger co‑regulation of the α‑linolenic acid and arachidonic acid metabolism than the soybean meal-fed group (P < 0.05). [Conclusion]  Protein supplementation enhances pathways related to energy metabolism and amino acid synthesis in worker bee hemolymph, thereby supporting royal jelly secretion in nurse bees. Among the protein sources evaluated, bee pollen provides superior integrated nutritional support than soybean meal, as demonstrated by the stronger enrichment of the lipid-metabolic and amino-acid biosynthetic pathways of bee-pollen fed bees. Notably, feeding bees rapeseed bee pollen resulted in greater modulation of essential fatty acid metabolism, lipid synthesis, and membrane-associated functions, indicating its suitability for precise adjustment of honeybee nutrition. Collectively, these findings provide a molecular basis for optimizing precision feeding strategies and formulating better artificial diets for honeybees.