The SMAP-18, an antimicrobial peptide derived from sheep myeloid antimicrobial peptide-29 (SMAP-29), exhibits a broad range of antibacterial activity against Gram-negative and positive bacteria. SMAP-18 showed a higher cell selectivity but lower cytotoxicity than SMAP-29. In addition, SMAP-18 penetrates the cells and kills bacteria without cell lysis. In contrast, SMAP-29 is targeting bacterial membrane, which induces a high cytotoxicity and low cell selectivity. Therefore, SMAP-18 can be a promising candidate for the development of new antibiotics. In this study, we designed and synthesized a series of SMAP-18 analogues, and then investigated their structures, antimicrobial activities, and bacterial killing mechanisms. The analogues include G→A mutants, N- and C-terminal truncated peptides of SMAP-18. Among them, G2,7,13A showed higher antimicrobial activity against Gram-negative bacteria and methicillin-resistant Staphylococcus aureus (MRSA). All peptides showed no helical propensity in H2O. However, in 50% trifluoroethanol, G7,G13→A mutants induced -helical structure. In addition, we determined 3D structure of SMAP-18 and G2,7,13A using 2D NMR spectroscopy to confirm the structural change by the mutation. Using fluorescence spectroscopy, we found that G7,G13→A mutants kills bacterial cells by damaging the cell membranes. These studies, suggest thet Gly substitution regulates -helix formation and affects on antimicrobial activity and bacterial killing mechanism. Therefore, both G7 and G13 play an important role in structure and antimicrobial activity of SMAP-18.