Cationic antimicrobial peptides (CAPs), including taxonomically diverse defensins, are innate defense molecules that display potent antimicrobial and immunomodulatory activities. Specific CAPs have also been shown to possess anticancer activities; however, their mechanisms of action are not well defined. Recently, the plant defensin NaD1 was shown to induce tumour cell lysis by directly binding to the plasma membrane phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2). The NaD1-lipid interaction was structurally defined by X-ray crystallography, with the defensin forming a dimer that binds PI(4,5)P2 via its cationic β2-β3 loops in a 'cationic grip' conformation. In this study, we show that human β-defensin 3 (HBD-3) contains a homologous β2-β3 loop that binds phosphoinositides. The binding of HBD-3 to PI(4,5)P2 was shown to be critical for mediating cytolysis of tumour cells, suggesting a conserved mechanism of action for defensins across diverse species. These data not only identify an evolutionary conservation of CAP structure and function for lipid binding, but also suggest that PIP-binding CAPs could be exploited for novel multifunction therapeutics.