Defensins belong to a diverse group of cationic antimicrobial peptides (CAPs) that are expressed throughout the plant and animal kingdoms as a first line of defense against invading microbial pathogens. An important mechanism of action for many defensins has been linked to membrane targeting and disruption. Selected plant defensins have been shown to form dimers that specifically bind the headgroups of membrane phospholipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidic acid (PA) through a ‘cationic grip’ conformation . Upon interaction with such lipids these defensins have been suggested to rupture cell membranes by forming oligomeric assemblies. We have previously determined the oligomeric structures of Nicotiana alata defensin 1 (NaD1) in complex with PIP21, and Nicotiana suaveolens defensin 7 (NsD7) in complex with PA 2 as well as PIP23. These three structures illustrate the different oligomeric arrangements that the defensins can adopt upon binding specific lipids. However, the molecular basis of the membrane disruption mechanism(s) of these defensin-lipid oligomers has not been defined. To gain further insight into the crucial underlying permeabilization mechanism of defensins we determined the structure of NaD1 in complex with PA to 2.5Å. The complex crystallized as a 20-meric unit comprising 10 NaD1 dimers and 14 PA bound with their acyl chais aligned in the same direction. The observed oligomer is flat, forming a defensin carpet-like arrangment that is strikingly different to the above mentioned defensin-lipid structures, which displayed long coil-like topologies. The overall topology of the NaD1–PA oligomer reveals a proposed membrane-attack configuration that could ultimately destabilize cell membranes through direct lipid sequestering and by inducing membrane curvature stress. Additionally, our functional assays supports the formation of this complex through a PA-only cooperative binding mechanism. These data provide structural evidence of the long-proposed carpet model of antimicrobial peptide membrane disruption.