IroN was recently identified in the extracellular pathogenic Escherichia coli strain CP9. In this study experimental evidence demonstrating that IroN mediates utilization of the siderophore enterobactin was obtained, thereby establishing IroN as a catecholate siderophore receptor. In a mouse model of ascending urinary tract infection the presence of iroN contributed significantly to CP9's ability to colonize the mouse bladder, kidneys, and urine, evidence that IroN is a urovirulence factor. However, growth in human urine ex vivo and adherence to uroepithelial cells in vitro were equivalent for an isogenic mutant deficient in IroN (CP82) and its wild-type parent (CP9). Taken together, these findings establish that IroN is a siderophore receptor and a urovirulence factor. However, uncertainty exists as to the mechanism(s) via which IroN contributes to urovirulence.