Poor proteolytic resistance is an urgent problem to be solved in the clinical application of antimicrobial peptides (AMPs), yet common solutions, such as complicated chemical modifications and utilization of d-amino acids, greatly increase the difficulty and cost of producing AMPs. In this work, a set of novel peptides was synthesized based on an antitrypsin/antichymotrypsin hydrolytic peptide structure unit (XYPX) n (X represents I, L, and V; Y represents R and K), which was designed using a systematic natural amino acid arrangement. Of these peptides, 16 with seven repeat units had the highest average selectivity index (GMSI = 99.07) for all of the Gram-negative bacteria tested and remained highly effective in combating Escherichia coli infection in vivo. Importantly, 16 also had dramatic resistance to a high concentration of trypsin/chymotrypsin hydrolysis and exerted bactericidal activity through a membrane-disruptive mechanism. Overall, these findings provide new approaches for the development of antiprotease hydrolytic peptides that target Gram-negative bacteria.