Antibiotic resistance is an increasingly severe health problem. Antimicrobial peptides (AMPs) are being developed in order to overcome this problem, due lower bacterial resistance. Polymyxin B is an AMP with bactericidal effect on Gram-negative bacteria due to its high affinity for lipopolysaccharide (LPS). The objective of this work was to unravel the polymyxin B mechanisms of LPS neutralization and bactericidal activity. Using dynamic light scattering, it was observed that polymyxin B induces LPS aggregation in a concentration-dependent manner. The peptide increases the surface charge of LPS and membrane model systems, as revealed by zeta-potential measurements. The higher zeta-potential variations were detected in the presence of the negatively charged POPG membranes. This higher interaction with negatively charged membranes, made of POPG, was followed at higher peptide concentration by membrane permeabilization. Also, for zwitterionic POPC membranes a higher membrane leakage was detected. The peptide promotion of LPS aggregation may be related with the clearance of LPS from the bloodstream, eventually by facilitating macrophage phagocytosis and/ or blocking the binding of LPS to its receptor. Our data indicate that polymyxin B mechanism of action at the molecular level involves a first step of electrostatic approach toward LPS; then, it may be internalized and bind to the bacterial phosphatidylglycerol-rich membrane leaflets, inducing leakage at higher peptide concentrations.