The aim of this study is to analyze the resistance function and mechanism of the putative ATP-binding cassette (ABC) transporter YgaD against polymyxin B in Bacillus subtilis.The interaction between the YgaD protein and the antimicrobial peptide polymyxin B was initially assessed using molecular docking and molecular dynamics simulation. Subsequently, resistance assays and intracellular polymyxin B content measurements were conducted on Bacillus subtilis with a knockout of the ygaD gene and on Escherichia coli with heterologous expression of the ygaD gene to validate the resistance function mediated by the YgaD protein and deduce its potential resistance mechanism.The results demonstrated that the YgaD protein could form stable complexes with polymyxin B and facilitated its efflux from bacterial cells, thereby reducing its intracellular accumulation and conferring resistance to polymyxin B.Our study revealed that YgaD regulates polymyxin B resistance in Bacillus subtilis through an efflux mechanism. These findings contribute to the understanding of microbial resistance mechanisms against antimicrobial peptides and provide a theoretical basis for the future design and development of antimicrobial drugs.Copyright © 2025 Elsevier Ltd. All rights reserved.