Extra Intestinal Pathogenic Escherichia coli (ExPEC) are the cause of a diverse spectrum of invasive infections in humans and animals, leading to urinary tract infections, meningitis or septicemia. In this paper, we focused our attention on the identification of the outer-membrane proteins of the pathogen in consideration of their important biological role and of their being potential targets for prophylactic and therapeutic intervention. To this aim, we generated a tolR mutant of the pathogenic IHE3034 strain, which spontaneously released a large quantity of OMVs in the culture supernatant. The vesicles were analysed by 2D-electrophoresis coupled to mass spectrometry. The analysis led to the identification of 100 proteins, most of which belonging to the outer membrane and periplasmic compartments. Interestingly, based on the genome sequences available in the current public database, seven of the identified proteins appear to be specific for pathogenic E. coli and enteric bacteria and therefore are potential targets for vaccine and drug development. Finally, we demonstrated that the cytolethal distending toxin, a toxin exclusively produced by pathogenic bacteria, is released in association with the vesicles, supporting the recently proposed role of bacterial vesicles in toxin delivery to host cells. Overall, our data demonstrated that OMVs represent an ideal tool to study Gram-negative periplasm and outer membrane compartments and to shed light on new mechanisms of bacterial pathogenesis.