Abstract Phospholipases are ubiquitous enzymes that cleave phospholipids, one major constituent of membranes. They are thus essential for many developmental processes, including male gamete development. In flowering plants, mutation of phospholipase NOT-LIKE-DAD (NLD) leads to peculiar defects in sexual reproduction. Indeed, pollination of a wild-type female with mutant pollen generates haploid embryos containing solely maternal genetic information. Contrary to previous reports NLD does not localize to cytosol and plasma membrane (PM) of sperm cells but to the pollen endo-plasma membrane (endo-PM), a specific membrane derived from the PM of the pollen vegetative cell that encircles the two sperm cells. Pharmacological approaches coupled with targeted mutagenesis revealed that lipid anchoring together with electrostatic interactions between membrane and NLD are involved in the attachment of NLD to this atypical endo-PM. Membrane surface-charge and anionic lipid bio-sensors indicated that phosphatidylinositol-4,5-bisphosphate (PIP(4,5)P 2 ) is enriched in the endo-PM as compared to the PM. Our results uncover a unique example of how membrane electrostatic properties can specify a unique polar domain (i.e. endo-PM), which is critical for plant reproduction and gamete formation.