Newly laid eggs of many insect species store maternal ecdysteroids as physiologically inactive phosphoric esters. In the silkworm Bombyx mori, we previously reported the presence of a specific enzyme, called ecdysteroid-phosphate phosphatase (EPPase), which catalyzes the dephosphorylation of ecdysteroid-phosphates to increase the amount of free ecdysteroids during early embryonic development. In this study, we demonstrated that (1) EPPase is found in the cytosol of yolk cells, (2) ecdysteroid-phosphates are localized in yolk granules, being bound to the yolk protein vitellin (Vn), and (3) Vn-bound ecdysteroid-phosphates are scarcely hydrolyzed by EPPase, although free ecdysteroid-phosphates are completely hydrolyzed by EPPase. Thus, we investigated the mechanism by which ecdysteroid-phosphates dissociate from the Vn-ecdysteroid-phosphate complex, and indicated that the acidification of yolk granules causes the dissociation of ecdysteroid-phosphates from the Vn-ecdysteroid-phosphate complex and thereby ecdysteroid-phosphates are released from yolk granules into the cytosol. Indeed, the presence of vacuolar-type proton-translocating ATPase in the membrane fraction of yolk granules was also verified by Western blot analysis. Our experiments revealed that Vn functions as a reservoir of maternal ovarian ecdysteroid-phosphates as well as a nutritional source during embryonic development. This is the first report showing the biochemical mechanism by which maternal Vn-bound ecdysteroid-phosphates function during early embryonic development.