Anopheles gambiae mosquitoes are the most important vectors of human malaria. The reproductive success of these mosquitoes relies on a single copulation event after which the majority of females become permanently refractory to further mating. This refractory behavior is at least partially mediated by the male-synthetized steroid hormone 20-hydroxyecdysone (20E), which is packaged together with other seminal secretions into a gelatinous mating plug and transferred to the female atrium during mating. In this study, we show that two 20E-regulated chymotrypsin-like serine proteases specifically expressed in the reproductive tract of An. gambiae females play an important role in modulating the female susceptibility to mating. Silencing these proteases by RNA interference impairs correct plug processing and slows down the release of the steroid hormone 20E from the mating plug. In turn, depleting one of these proteases, the Mating Regulated Atrial Protease 1 (MatRAP1), reduces female refractoriness to further copulation, so that a significant proportion of females mate again. Microscopy analysis reveals that MatRAP1 is localized on a previously undetected peritrophic matrix-like structure surrounding the mating plug. These data provide novel insight into the molecular mechanisms shaping the post-mating biology of these important malaria vectors.