Insect seminal fluid proteins are powerful modulators of many aspects of female physiology and behaviour including longevity, egg production, sperm storage, and remating. The crucial role of these proteins in reproduction makes them promising targets for developing tools aimed at reducing the population sizes of vectors of disease. In the malaria mosquito Anopheles gambiae, seminal secretions produced by the male accessory glands (MAGs) are transferred to females in the form of a coagulated mass called the mating plug. The potential of seminal fluid proteins as tools for mosquito control demands that we improve our limited understanding of the composition and function of the plug. Here, we show that the plug is a key determinant of An. gambiae reproductive success. We uncover the composition of the plug and demonstrate it is formed through the cross-linking of seminal proteins mediated by a MAG-specific transglutaminase (TGase), a mechanism remarkably similar to mammalian semen coagulation. Interfering with TGase expression in males inhibits plug formation and transfer, and prevents females from storing sperm with obvious consequences for fertility. Moreover, we show that the MAG-specific TGase is restricted to the anopheline lineage, where it functions to promote sperm storage rather than as a mechanical barrier to re-insemination. Taken together, these data represent a major advance in our understanding of the factors shaping Anopheles reproductive biology.