Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus-oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.