Aims Emergence and rapid dissemination of antibiotic-resistant bacteria is becoming a severe problem to public health. The search for antimicrobial substitutes for antibiotics is necessary. Lactoferricin B like peptide (LBLP) is a 23-mer antimicrobial peptide (AMP), derived from the big centipede Scolopendra subspinipes mutilans. Although its antifungal effect and its mechanism have been reported, the antibacterial activity has not yet been elucidated. Method and Results In this study, we investigated antibacterial activity of LBLP and its mode of action. LBLP showed potent antibacterial effect against pathogenic bacteria Escherichia coli and did not show haemolytic activity against human erythrocyte. The general antimicrobial mechanism of AMP is to disrupt the cell membrane, however, LBLP exerted its antibacterial activity by causing apoptosis-like death through reactive oxygen species (ROS) generation. LBLP-treated E. coli cells exhibited hallmarks of apoptosis, such as membrane depolarization, DNA fragmentation, caspase-like protein activation and phosphatidylserine exposure. These apoptotic features were attenuated by pretreatment of NAC, a representative ROS scavenger. Conclusions These results demonstrate that LBLP exerted its antibacterial activity by generating ROS and inducing apoptosis-like death in E. coli. LBLP is not membrane destructive per se, but essentially a metabolic inhibitor. Significance and Impact of the Study Lactoferricin B like peptide is potential candidate to replace conventional antibiotics that are less effective because of its unique properties.