<p>Natural antimicrobial peptides frequently exhibit characteristic terminal features, such as a free amino group at the N-terminus and C-terminal amidation, that are believed to enhance antimicrobial performance. However, the molecular rationale underlying this evolutionarily favored structural motif remains insufficiently understood. In this study, we systematically investigated the influence of terminal capping on Mastoparan C (MPC), a representative helical amphipathic peptide, by combining structural and biological evaluation with molecular dynamics (MD) simulations. Our findings indicate that this common structural motif of terminal modifications in antimicrobial peptides is a strategic adaptation to optimize functional performance, enhancing the ability of these peptides to effectively disrupt microbial membranes while maintaining selectivity. This study not only provides insight into the evolutionary design of antimicrobial peptides but also guides the development of synthetic analogs for therapeutic applications. Impact of terminal capping design on antimicrobial peptide: Three analogs of Mastoparan C (<b>H-MPC-NH2</b>) were prepared with the same sequence but different terminal capping groups. We demonstrated that the free amino group at N-terminal and amidated C-terminal gave the best in improving antibacterial potency and selectivity, thus providing insights into the reason this terminal capping design is popular in antimicrobial peptides.</p> Graphical abstract <p></p>