Uropathogenic Escherichia coli (UPEC) is a leading etiological agent of bacteremia in humans. Virulence mechanisms of UPEC in the context of urinary tract infections have been subjected to extensive research. However, understanding of the fitness mechanisms used by UPEC during bacteremia and systemic infection is limited. A forward genetic screen was utilized to detect transposon insertion mutants with fitness defects during colonization of mouse spleens. An inoculum comprised of 360,000 transposon mutants in the UPEC strain CFT073, cultured from the blood of a patient with pyelonephritis, was used to inoculate mice intravenously. Transposon insertion sites in the inoculum (input) and bacteria colonizing the spleen (output) were identified using high-throughput sequencing of transposon-chromosome junctions. Using frequencies of representation of each insertion mutant in the input and output samples, 242 candidate fitness genes were identified. Co-infection experiments with each of 11 defined mutants and the wild-type strain demonstrated that 82% (9 of 11) of the tested candidate fitness genes were required for optimal fitness in a mouse model of systemic infection. Genes involved in biosynthesis of poly-N-acetyl glucosamine (pgaABCD), major and minor pilin of a type IV pilus (c2394 and c2395), oligopeptide uptake periplasmic-binding protein (oppA), sensitive to antimicrobial peptides (sapABCDF), putative outer membrane receptor (yddB), zinc metallopeptidase (pqqL), a shikimate pathway gene (c1220) and autotransporter serine proteases (pic and vat) were further characterized. Here, we report the first genome-wide identification of genes that contribute to fitness in UPEC during systemic infection in a mammalian host. These fitness factors may represent targets for developing novel therapeutics against UPEC.