<p>The antimicrobial peptides, such as host immune system-derived human bactericidal/permeability-increasing protein (hBPI), are the potential drugs for antibiotic-resistant Gram-negative bacterial infection. However, whether the purified chimeric hBPI₂₃-Fcγ protein has bactericidal activity against drug-resistant Gram-negative bacteria (GNB) and the relevant mechanisms have not been fully elucidated. In this study, the chimeric hBPI₂₃-Fcγ protein, which consisting of the functional N terminus of BPI and Fcγ1, were expressed and purified in a lab-scale. The chimeric hBPI₂₃-Fcγ protein showed longer half-life up to 148.2&#xa0;min in vivo. The hBPI₂₃-Fcγ protein also showed significant bactericidal activity against standard and clinically isolated drug-resistant <i>Acinetobacter baumannii</i> (<i>A. baumannii)</i> and <i>Escherichia coli</i> (<i>E. coli</i>). In addition, the hBPI₂₃-Fcγ protein markedly decreased biofilm formation, neutralized bacterial lipopolysaccharides (endotoxin) and enhanced the opsonization of phagocytes, as well as significantly improved the survival rate of minimal lethal dose (MLD) of drug-resistant <i>E. coli</i> -infected mice. These results indicate that the BPI₂₃-Fcγ protein protected mice from drug-resistant GNB infection not only by direct bactericidal effect, but also by promoting opsonophagocytosis of macrophages. In conclusion, the chimeric BPI₂₃-Fcγ protein may be as a promising candidate of non-antibiotic biological agent for drug-resistant GNB infection.</p> Graphical Abstract <p></p>