Ostreolysin A6, a 15 kDa protein produced by the oyster mushroom (Pleurotus ostreatus), binds to membrane cholesterol/sphingomyelin domains and, together with a larger protein partner pleurotolysin B (PlyB) co-produced by the same organism, forms 13-meric transmembrane pore complexes. Further, OlyA6 can bind even 1000-times more strongly to an insect-specific membrane sphingolipid ceramide phosphoethanolamine (CPE) and, in concert with PlyB, exert potent and selective insecticidal activity against two economically important coleopteran pests: the Colorado potato beetle and the western corn rootworm (WCR). We have used cryo-electron microscopy to further explore the mechanism of membrane interaction of OlyA6/PlyB complexes with lipid vesicles composed of artificial lipids containing CPE, and with WCR brush border membrane vesicles. In both lipid preparations, the formation of multimeric transmembrane pores, similar to those described on cholesterol/sphingomyelin membranes, was observed. We have also analysed the histological alterations of columnar epithelium comprising the midgut wall of WCR larvae fed with OlyA6/PlyB mixture, including vacuolisation of cytoplasm, swelling of apical cell surface into the gut lumen, and delamination of basal lamina underlying the epithelium. Our combined results strongly suggest that the molecular mechanism of insecticidal action of OlyA6/PlyB arises from the specific interaction of OlyA6 with the invertebrate-specific membrane lipid receptor, and consequent formation of transmembrane pores in the insect midgut. This mode of membrane binding is different from those described for similar aegerolysin-based complexes of bacterial origin (e.g. Cry34Ab1/Cry35Ab1), or other B. thuringiensis Cry toxins, which associate with protein receptors.