Royal jelly (RJ) is a proteinaceous secretion of the hypopharyngeal glands (HGs) in the head of honeybee workers. It is a critical food for queen bees and young larvae that decides the fate of fertilized eggs in developing into either queen bees or worker bees during the early larval stages. RJ is also widely used in humans for health promotion as agent, such as antibacterial, antioxidant, and antiaging properties. To increase RJ yields, a stock of high RJ producing honeybees (RJBs) has genetically selected from Italian honeybees (ITBs) in China since 1980s. To date, one colony of RJBs can produce more than 10 kg of RJ per year, a yield that is 10-times greater than that produced by a colony of ITBs. To elucidate the mechanism of the enforced gland performance in producing RJ in RJBs, the spatio-temporal HG proteomes of newly emerged bees, nurse bees, and forager bees, were compared between the ITBs and RJBs. Proteins in the critical pathways that are implicated in the secretory activity of RJ in HGs are validated biochemically and biologically by manipulating the NBs into extended nursing periods and the FBs to revert into NBs. This will provide a novel mechanistic insight into the HGs achieving an enhanced biological mission of producing the valuable bee-product, RJ.