In colorectal cancer, adenomatous polyposis coli (APC) interacts with Rho guanine-nucleotide-exchange factor 4 (Asef), thereby stimulating aberrant colorectal-cancer-cell migration. Consequently, the APC-Asef interaction represents a promising therapeutic target for mitigating colorectal-cancer migration. In this study, we adopted the rational-design strategy involving the introduction of intramolecular hydrogen bonds and optimization of the lipophilic substituents to improve the binding affinities of peptides, leading to the discovery of MAI-400, the best inhibitor of the APC-Asef interaction known to date ( Kd = 0.012 μM, IC50 = 0.25 μM). Comprehensive evaluation of MAI-400 by biochemical and biophysical assays revealed the formation and effect of an intramolecular hydrogen bond. A cell-based assay showed MAI-400 efficiently blocking the APC-Asef interaction in a dose-dependent manner. Therefore, our study provides a best-in-class inhibitor, MAI-400, based on the rational drug design and structural validation, that can effectively inhibit the APC-Asef interaction.