Fertilization is fundamental for sexual reproduction, yet its molecular mechanisms are poorly understood. We found that an oocyte-expressed Ly6/uPAR protein, which we call Bouncer, is a crucial fertilization factor in zebrafish. Membrane-bound Bouncer mediates sperm-egg binding and is thus essential for sperm entry into the egg. Remarkably, Bouncer not only is required for sperm-egg interaction but is also sufficient to allow cross-species fertilization between zebrafish and medaka, two fish species that diverged more than 200 million years ago. Our study thus identifies Bouncer as a key determinant of species-specific fertilization in fish. Bouncer's closest homolog in tetrapods, SPACA4, is restricted to the male germline in internally fertilizing vertebrates, which suggests that our findings in fish have relevance to human biology.