Cationic antimicrobial peptides have attracted increasing attention as a novel class of antibiotics to treat infectious diseases caused by pathogenic bacteria. However, susceptibility to protease is a shortcoming in their development. Cyclization is one approach to increase the proteolytic resistance of peptides. Therefore, to improve the proteolytic resistance of Polybia-MPI, we have synthesized the MPI cyclic analogs C-MPI-1 (i-to-i+4) and C-MPI-2 (i-to-i+6) by copper(I)-catalyzed azide-alkyne cycloaddition. Compared with MPI, C-MPI-1 displayed sustained antimicrobial activity and had enhanced anti-trypsin resistance, while C-MPI-2 displayed no antimicrobial activity. The relationship between peptide structure and bioactivity was further investigated by probing the secondary structure of the peptides by circular dichroism. This showed that C-MPI-1 adopted an α-helical structure in aqueous solution and, interestingly, had increased α-helical conformation in 30 mM sodium dodecyl sulfate and 50% trifluoroethyl alcohol compared with MPI. C-MPI-2 that was not α-helical in structure, suggesting that the propensity for α-helix conformation may play an important role in cyclic peptide design. In addition, scanning electron microscopy, propidium iodide uptake, and membrane permeabilization assays indicated that MPI and the optimized analog C-MPI-1 had membrane-active action modes, indicating that the peptides would not be susceptible to conventional resistance mechanisms. Our study provides additional insight into the influence of intramolecular cyclization at various positions on peptide structure and biological activity. In conclusion, the design and synthesis of cyclic analogs via click chemistry offer a new strategy for the development of stable antimicrobial agents. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.