BackgroundExtracellular vesicles (EVs) from Gram-positive bacteria have gained considerable importance as a novel transport system of virulence factors in host–pathogen interactions.Bacillus cereusis a Gram-positive human pathogen, causing gastrointestinal toxemia as well as local and systemic infections. The pathogenicity of enteropathogenicB. cereushas been linked to a collection of virulence factors and exotoxins. Nevertheless, the exact mechanism of virulence factor secretion and delivery to target cells is poorly understood.ResultsHere, we investigate the production and characterization of enterotoxin-associated EVs from the enteropathogenicB. cereusstrain NVH0075-95 by using a proteomics approach and studied their interaction with human host cells in vitro. For the first time, comprehensive analyses ofB. cereusEV proteins revealed virulence-associated factors, such as sphingomyelinase, phospholipase C, and the three-component enterotoxin Nhe. The detection of Nhe subunits was confirmed by immunoblotting, showing that the low abundant subunit NheC was exclusively detected in EVs as compared to vesicle-free supernatant. Cholesterol-dependent fusion and predominantly dynamin-mediated endocytosis ofB. cereusEVs with the plasma membrane of intestinal epithelial Caco2 cells represent entry routes for delivery of Nhe components to host cells, which was assessed by confocal microscopy and finally led to delayed cytotoxicity. Furthermore, we could show thatB. cereusEVs elicit an inflammatory response in human monocytes and contribute to erythrocyte lysis via a cooperative interaction of enterotoxin Nhe and sphingomyelinase.ConclusionOur results provide insights into the interaction of EVs fromB. cereuswith human host cells and add a new layer of complexity to our understanding of multicomponent enterotoxin assembly, offering new opportunities to decipher molecular processes involved in disease development.Video AbstractSupplementary InformationThe online version contains supplementary material available at 10.1186/s12964-023-01132-1.