Plant defensins are small innate immune peptides that often exhibit broad-spectrum antifungal activity.  NaD1 is an antifungal class II defensin of the Solanaceae family that has also recently been shown to exhibit selective cytotoxic activity towards mammalian tumour cells and represents a promising candidate for the development of anticancer therapeutics.  NaD1 acts through a novel phosphatidylinositol 4,5-bisphosphate (PIP2)-binding mechanism involving PIP2-mediated oligomerization of NaD1 leading to target membrane destabilization. The class II tomato defensin, TPP3, is a related yet poorly characterized homologue of NaD1. Using several biological and biophysical approaches, we show that TPP3 shares structural and functional features with NaD1, indicating an evolutionarily conserved lipid-binding mechanism of action within this class of defensin. TPP3 exhibited comparable antitumour activity to NaD1, which was shown to be dependent on target cell membrane PIP2 to elicit efficient membrane lysis. Structural determination by X-ray crystallography revealed striking similarities between TPP3 and NaD1, with TPP3 adopting the lipid-binding ‘cationic grip’ dimer conformation of NaD1, indicating that TPP3 is likely to form an oligomeric complex in the presence of its ligand, PIP2. These findings contribute to our broadening understanding of class II solanaceous plant defensins and their therapeutic potential as anticancer molecules.