In species in which females mate repeatedly, males can adopt several strategies to reduce the risk of sperm competition with future males. Mated females of West Indian sweet potato weevil Euscepes postfasciatus (Coleoptera) temporarily become unreceptive to subsequent mating, however the physiological mechanism underlying this has not been clarified. To elucidate this mechanism, we investigated the effects of male-derived substances on the inhibition of mating receptivity of virgin females by injecting the substances into their abdomens. The length of time from injection to mating in virgin females was significantly longer in females injected with accessory gland B solution than in those injected with control solution. In this study, we showed that mated females have a long refractory period because males of E. postfasciatus inhibit female remating by using seminal fluid substances to ensure their paternity.