In amphibians, the presence in the fertilization medium of diffusible components from the jelly coat, called diffusible factors, is indispensable. However, few studies of anuran species have characterized the biomolecule capable of inducing the acrosome reaction. Our previous studies of a species of anuran amphibian (Rhinella arenarum) from the Andean Yungas of northwestern Argentina demonstrated that uterine oocytes that contain the jelly coat lose fertilizability in the insemination medium depending on washing time and washing medium. Such loss can be reversed by the addition of all the proteins and glycoproteins from the diffusible factor (DF), an effect that reaches a maximum with the addition of exogenous Ca²⁺. In the present study, we employed an experimental model to demonstrate the biological effect of DF and of four purified protein fractions from DF on conditioning coelomic oocytes. Our experiments demonstrated that only purified fraction 2 of DF, which contains only a 74 KDa diffusible glycoprotein (gp74), caused characteristic modifications on the surface of the conditioned coelomic oocytes, identical to the ones generated by the acrosomal lysine solution. These modifications of the oocytes did not occur in the absence of sperm in the medium. Furthermore, previous treatment of a sperm suspension with gp74 and later incubation of the supernatant obtained from this pretreatment with the conditioned coelomic oocytes caused the same lytic effect on the oocyte surface, confirming that gp74 participates by inducing the release of the acrosomal content in the insemination medium. These results are a new contribution to the knowledge on the fertilization of this anuran species and allow us to continue with the study of the gp74 receptor in the spermatozoon.