High royal jelly producing bees (RJBs), a stock of honeybees selected from Italian bees (ITBs), have developed a stronger worker reproductive investment via alloparental care that leads to colony-level RJB syndrome. However, the molecular basis of extreme reproductive investment of RJBs is not yet clear. In this study, activity-based proteome profiling was used to compare specific molecular changes in hypopharyngeal glands (HGs) between ITBs and RJBs that may provide new insights into the molecular basis of colony-level RJB syndrome.