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Extraintestinal pathogenic Escherichia coli (ExPEC) are a common cause of disease in both mammals and birds. A vaccine to prevent such infections would be desirable given the increasing antibiotic resistance of these bacteria. We have determined the genome sequence of ExPEC IHE3034 (ST95) isolated from a case of neonatal meningitis and compared this to available genome sequences of other ExPEC strains and a few nonpathogenic E. coli. We found 19 genomic islands present in the genome of IHE3034, which are absent in the nonpathogenic E. coli isolates. By using subtractive reverse vaccinology we identified 230 antigens present in ExPEC but absent (or present with low similarity) in nonpathogenic strains. Nine antigens were protective in a mouse challenge model. Some of them were also present in other pathogenic non-ExPEC strains, suggesting that a broadly protective E. coli vaccine may be possible. The gene encoding the most protective antigen was detected in most of the E. coli isolates, highly conserved in sequence and found to be exported by a type II secretion system which seems to be nonfunctional in nonpathogenic strains.