Infections due to extraintestinal pathogenic Escherichia coli (ExPEC) are common in humans and animals and include urinary tract infections (from uropathogenic E. coli [UPEC]), septicemia, and wound infections. These infections result in significant morbidity and mortality and in high health care costs. In view of the increasing number of ExPEC infections and the ever-growing antibiotic resistance capability of ExPEC isolates, preventive measures such as an effective vaccine against ExPEC are desirable. An ExPEC vaccine may be cost-effective for select patient groups. Previous vaccine candidates consisted of single target proteins or whole ExPEC cells. Here we describe a subunit vaccine against ExPEC which is based on immunodominant epitopes of the virulence-associated ExPEC proteins FyuA, IroN, ChuA, IreA, Iha, and Usp. Using a novel approach of computer-aided design, two completely artificial genes were created, both encoding eight peptide domains derived from these ExPEC proteins. The recombinant expression of these two genes resulted in a protein vaccine directed against ExPEC but not against commensal E. coli of the gut flora. In mice, the vaccine was highly immunogenic, eliciting both strong humoral and cellular immune responses. Nasal application resulted in high secretory immunoglobulin A (sIgA) production, which was detectable on the mucosal surface of the urogenital tract. Finally, it conveyed protection, as shown by a significant reduction of bacterial load in a mouse model of ExPEC peritonitis. This study provides evidence that a novel vaccine design encompassing distinct epitopes of virulence-associated ExPEC proteins may represent a means for providing a protective and pathogen-specific vaccine.