The mutant of Ras protein with serine to asparagine mutation at residue 17 (Ras-17N) is known to interfere with the signaling function of the wild-type Ras protein by sequestering its guanine-nucleotide exchange factors (GEFs). The similar mutant of another Ras family protein Rap1 (Rap1-17N) fails to effectively interfere with the interaction between the wild-type Rap1 and one of its GEFs, C3G, in vitro. In the present study, we have attempted to isolate Rap1 mutants with increased affinity for C3G using random mutagenesis and yeast two-hybrid screening. Based on the pattern of mutations found among these mutants, we could design a potent C3G-binder, named Rap1-AGE, harboring mutations in three sites (17A, 29G, and 117E). The association of Rap1-AGE with C3G in the cells was confirmed by co-immunoprecipitation experiments. The ability of Rap1-AGE to inhibit C3G-mediated Rap1-activation and cell spreading was also demonstrated. On the other hand, Rap1 activation mediated by two other GEFs, Epac and smgGDS, was not inhibited by Rap1-AGE. These results suggest that Rap1-AGE acts as a dominant interfering factor against C3G and serves as a useful tool in analyzing the roles of C3G-Rap1 signaling pathway in various biological processes.