A structurally nanoengineered antimicrobial polypeptide consisting of lysine and valine residues is a new class of antimicrobial agent with superior antibacterial activity against multidrug-resistant bacteria and low toxicity toward mammalian cells. Utilizing coarse-grained models, we studied the interactions of microbial cytoplasmic membranes with polypeptides of either (K2V1)5 (star-KV) or CM15 (star-CM15). Our computational results verify the low toxicity of polypeptides of (K2V1)5 toward the dipalmitoyl phosphatidylcholine bilayer. This low toxicity is demonstrated to originate from weakened hydrophobicity combined with its random coil conformation for (K2V1)5 because of the highly abundant valine residues, compared with the typical antimicrobial peptides, such as CM15. In the interactions with a palmitoyl-oleoyl-phosphatidylethanolamine/palmitoyl-oleoyl-phosphatidylglycerol bilayer, star-KV has greater ability in phase separation and generation of phase boundary defects not only in lipid redistribution but also in lateral dynamic movements, although both star-KV and star-CM15 can extract the phosphatidylglycerol lipids and purify the phosphatidylethanolamine lipids into continuum domains. We suggest that the polypeptide of (K2V1)5 can nondisruptively kill bacteria by hampering bacterial metabolism through reorganizing lipid domain distribution and simultaneously "freezing" lipid movement.