Cationic antimicrobial peptides (CAPs) such as defensins are ubiquitously found innate immune molecules that often exhibit broad activity against microbial pathogens and mammalian tumor cells. Many CAPs act at the plasma membrane of cells leading to membrane destabilization and permeabilization. Here we describe a novel cell lysis mechanism for fungal and tumor cells by plant and human defensins that act via direct binding to the plasma membrane phospholipids phosphatidylinositol 4,5-bisphosphate (PIP2) or phosphatidic acid (PA). We have determined the crystal structures of the plant defensins NaD1 and NsD7 in complex with PIP and PA, respectively, revealing distinct oligomeric arrangements. Both NaD1 and NsD7 form dimers that cooperatively bind the anionic head groups of PIP2 or PA via unique “cationic grip” configurations and assemble into oligomeric fibrils (1,2). Site-directed mutagenesis of NaD1and NsD7 confirms that phospholipid binding and oligomerization are important for fungal and tumor cell permeabilization. We have shown that human beta-defensins 2 and 3 also use similar mechanisms for cell lysis. These observations identify a conserved innate recognition system by defensins for direct binding of phospholipid that permeabilize cells via a novel membrane disrupting mechanism.