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Mated queen honeybees (Apis mellifera) store viable sperm in their spermathecae for egg fertilization throughout their lifespan, which can extend for several years. However, the mechanisms underlying this remarkable ability for longterm sperm storage remain unclear. It is known that the molecular basis for sperm survival in queen bees' spermathecae is associated with minimized energy metabolism and reduced production of reactive oxygen species (ROS). However, the factors affecting sperm motility under these conditions have not yet been fully elucidated. This study presents novel evidence for the presence of a sperm-binding protein, Amwaprin, in both the seminal and spermathecal fluids of honeybees. RT-qPCR analysis and immunoassays revealed that Amwaprin is primarily expressed in the testes of drones and the spermathecae of queen honeybees. Furthermore, binding assays revealed that the reduction of Amwaprin on the sperm surface led to increased H2O2 production, more circular sperm morphology, increased sperm motility, and decreased sperm viability. These findings suggest that Amwaprin binding reduces the motility of sperm stored in queen bees' spermathecae, thereby minimizing energy expenditure and ROS production, which ultimately promotes long-term sperm survival. [ABSTRACT FROM AUTHOR]