Necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) are secreted by several phytopathogenic micro-organisms. They trigger leaf necrosis and immunity-associated responses in various dicotyledonous plants. We identified glycosylinositol phosphorylceramides (GIPC), a major class of plant sphingolipids, as target molecules for NLP binding to plasma membranes. X-ray crystallography revealed that NLP forms complexes with terminal hexose moieties. NLP binding to GIPC head groups induces several conformational changes within the toxin that precedes membrane attachment and host cell lysis. This study unveils early steps of NLP cytolysin action and explains why dicot plants are NLP toxin-sensitive whereas monocot plants are not.