Internal fertilization requires live sperm to be transferred from male to female before egg fertilization. Both males and females assist the insemination process by providing sperm with glandular secretions, which have been inferred to contain subsets of proteins that maintain sperm viability. Here we show that in the honeybee (Apis mellifera) secretions of the male accessory glands, the major contributors towards seminal fluid, enhance sperm survival. We further demonstrate that the protein fraction of the male accessory gland secretion is indeed important for achieving the maximal effect on sperm survival. After sperm storage, the queens also provide sperm with secretions from spermathecal glands and we show that these secretions have a comparable positive effect on sperm viability. SDS gels show that the proteomic profiles of accessory gland secretion and spermathecal fluid secretion hardly overlap, which suggests that males and females use different proteins to enhance sperm viability during, respectively, ejaculation and final sperm storage.