Naturally occurring cyclic antimicrobial peptides (AMPs) such as tyrocidine A (Tyrc A) and gramicidin S (GS) are appealing targets for the development of novel antibiotics. However, their therapeutic potentials are limited by undesired hemolytic activity and relatively poor activity against Gram-negative bacteria. Inspired by polycationic lipopeptide polymyxin B (PMB), the so called 'last-resort' antibiotic for the treatment of infections caused by multidrug-resistant Gram-negative bacteria, we synthesized and biologically evaluated a series of polycationic analogues derived from Tyrc A. We were able to obtain peptide 8 that possesses 5 positive charges exhibiting potent activities against both Gram-negative and Gram-positive bacteria along with totally diminished hemolytic activity. Intriguingly, antibacterial mechanism studies revealed that, rather than the 'pore forming' model that possessed by Tyrc A, peptide 8 likely diffuses membrane in a 'detergent-like' manner. Furthermore, when treating mice with peritonitis-sepsis, peptide 8 showed excellent antibacterial and anti-inflammatory activities in vivo.