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Membrane fusion is an essential step in the encounter of two nuclei from sex cells--sperm and egg--in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that sperm-egg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9⁻/⁻ eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9⁻/⁻ eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9⁻/⁻ eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.