Endogenous antimicrobial peptides (AMPs) are evolutionarily ancient molecular factors of innate immunity that play a key role in host defense. The study of the diversity of animal defense peptides has important applications in the context of the growing global antimicrobial resistance. In this study using a transcriptome mining approach, we found three novel thanatin-like β-hairpin AMPs in the bean bug , named Rip-2, Rip-3, and Rip-4. The peptides were expressed in the bacterial system, and their antimicrobial activities were evaluated both in vitro and in vivo. Homologs of the discovered AMPs are widely distributed among different members of the infraorder Pentatomomorpha. Rip-2 was shown to have the most similar structure and LptA-targeting mechanism of action to those of thanatin, but the former peptides demonstrated a higher activity against key Gram-negative ESKAPE pathogens and also displayed a significant efficacy in a lethal model of septicemia caused by in mice at daily doses greater than 5 mg/kg. In contrast, Rip-3 and Rip-4 peptides caused bacterial membrane damage, did not induce bacterial resistance, and exhibited a strong selectivity against and spp. This study extends the knowledge of the structure and functions of insect host defense AMPs. Each of the novel β-hairpin peptides has a potential to be a template for the development of selective antibiotic drugs.