This report clarifies the interaction of surface charge-modified gold nanoparticles (AuNPs) with phospholipid membranes, which is helpful to understand the antibacterial mechanism of positive charge-modified AuNPs to Gram-negative bacteria. Although the simulated bacterial cell membranes as a whole are negatively charged, the local electrostatic repulsive interaction between the positive charge-coated AuNPs and the small-sized flexible cationic head group of dioleyl phosphatidylethanolamine molecules induces the phase transformation of the simulated bacterial cell membranes from a lamellar to an inverted hexagonal phase. Transmembrane pores with a diameter of about 3.0 nm in the inverted hexagonal structure would result in the destruction of cell membrane function. Such an interaction of positive charge-modified AuNPs with the membrane mimics provides a promising route to develop new antibacterial agents by modifying positive charges on the surface of nanoparticles.