Background: Strains of Escherichia coli cause a wide variety of intestinal and extra-intestinal diseases in both humans and animals, and are also often found in healthy individuals or the environment. Broadly, a strong phylogenetic relationship exists that distinguishes most E. coli causing intestinal disease from those that cause extra-intestinal disease, however, isolates within a recently described subclass of Extra-Intestinal Pathogenic E. coli (ExPEC), termed endometrial pathogenic E. coli, tend to be phylogenetically distant from the vast majority of characterised ExPECs, and more closely related to human intestinal pathogens. In this work, we investigate the genetic basis for ExPEC infection in the prototypic endometrial pathogenic E. coli strain MS499. Results: By investigating the genome of MS499 in comparison with a range of other E. coli sequences, we have discovered that this bacterium has acquired substantial lengths of DNA which encode factors more usually associated with ExPECs and less frequently found in the phylogroup relatives of MS499. Many of these acquired factors, including several iron acquisition systems and a virulence plasmid similar to that found in several ExPECs such as APEC O1 and the neonatal meningitis E. coli S88, play characterised roles in a variety of typical ExPEC infections and appear to have been acquired recently by the evolutionary lineage leading to MS499. Conclusions: Taking advantage of the phylogenetic relationship we describe between MS499 and several other closely related E. coli isolates from across the globe, we propose a step-wise evolution of a novel clade of sequence type 453 ExPECs within phylogroup B1, involving the recruitment of ExPEC virulence factors into the genome of an ancestrally non-extraintestinal E. coli, which has repurposed this lineage with the capacity to cause extraintestinal disease. These data reveal the genetic components which may be involved in this phenotype switching, and argue that horizontal gene exchange may be a key factor in the emergence of novel lineages of ExPECs.