Helicobacter pylori (H. pylori) infection is highly prevalent, and has developed antimicrobial resistance to virtually all existing antibiotics. Currently, treatment of H. pylori infection (involving proton pump inhibitors and broad-spectrum antibiotics) is suboptimal, with high failure rates. Thus, there is a pressing need to develop new anti-H. pylori therapies. Cbf-K, a cathelicidin-like antimicrobial peptide, presented broad antimicrobial activity during our previous research. This study further evaluated the therapeutic potential and the mode of action underlying Cbf-Kagainst clarithromycin- and amoxicillin-resistant H. pylori SS1. The MIC and MBC of Cbf-Kagainst the tested H. pylori were 16 and 32 μg/ml, respectively, and its killing kinetics was time-dependent, reflecting the thorough elimination of drug-resistant bacteria within 24 h. This peptide also protected H. pylori-infected gastric epithelial cells (GES-1) from death by reducing the cell supernatant and intracellular bacterial counts by 1.9 and 2.9-logunits, respectively. These data indicated the powerful antimicrobial effects of Cbf-Kin vitro. Meanwhile, notable antimicrobial activity in the mouse gastritis model was observed, with decreasing bacterial counts by 3.9-logunits in stomach tissues and Cbf-Kcould effectively suppress the secretion of inflammatory cytokine IL-8. For its mode of action, Cbf-Knot only neutralized the negative potential and increased the membrane uptake of NPN and PI by 78.5% and 85.1%, respectively, but also bound to genomic DNA, which in turn downregulated the expression of adhesion genes (alpA and alpB) and virulence gene (cagA), indicating its effective activities on membrane disruption, DNA-binding and gene expression. The data above demonstrated that Cbf-Kpossessed effective antimicrobial and anti-inflammatory activities and downregulated the expression of adhesion- and cytotoxin-associated genes of drug-resistant H. pylori SS1, making it a potential candidate for anti-infective therapy.