Human α-defensin 5 (HD5) is a broad-spectrum antibacterial peptide produced by small intestinal Paneth cells. Despite considerable experimental evidence for the correlation between bacterial membrane destruction and the antibacterial activity of HD5, its membrane disruption mechanism remains unclear. Using all-atom molecular dynamics simulations and molecular mechanics Poisson-Boltzmann surface area analysis, we demonstrate the membrane disruption mechanism of HD5 based on the intrinsic binding of HD5 to Gram-negative (GN) bacterial inner membrane. It was found that both monomer and dimer forms of HD5 bind to the surface of the GN membrane rather than embedding in the hydrophobic core region of the bilayer. Regardless of the form of HD5, the peptide orientated itself similarly on the membrane surface with an inward-pointing electric dipole moment and an outward-pointing hydrophobic dipole moment. We investigated its possible membrane disruption mechanisms and determined that anionic lipid clustering is a plausible mode of action for HD5. Relative binding free energy analysis revealed that electrostatic interactions play a major role in this mechanism. Our findings shed light on the biophysical phenomena of HD5-GN membrane binding and suggest a possible membrane disruption mechanism for HD5. This analysis of the fundamental binding properties of the monomeric HD5-GN membrane complex provides a useful guide for defensin-derived antibiotic design.