Escherichia coli O157:H7 is a non-invasive Shiga toxin (Stx)-producing pathogen that causes gastroenteritis and hemolytic uremic syndrome characterized by acute kidney injury. Stx binding to cells induces release of extracellular vesicles (EVs) containing the toxin. This study aimed to demonstrate the lethal effects of toxin-positive vesicles compared to free toxin, and the importance of the Stx receptor, globotriaosylceramide (Gb3), for these effects.