The C domain of eRF1 interacts with the C domain of eRF3, and the binding of both factors is essential for fast kinetics of the termination of protein translation. Analysis by computational simulation demonstrated that several peptides involved in Eo-eRF1/Eo-eRF3 interaction directly. Among these peptides, the two motifs GVEDT and GFGG were highly conserved, while the fragment aa338-346 of Eo-eRF1a/b was variable. In additional, I290 and D293 of Eo-eRF1 were also highly conserved. By the site-directed mutagenesis and pull-down analysis, the amino acid D293 in Eo-eRF1bC domain was conformed playing an important role in eRF1-eRF3 interaction. Eo-eRF1a and Eo-eRF1b may select different manners to interact with Eo-eRF3. These studies contribute to the better understanding the mode of eRF1-eRF3 interaction.