In E. coli, the small regulatory non-coding RNA RyhB and the global ferric uptake regulator (Fur) mediate iron acquisition and storage control. Iron is both essential and potentially toxic for most living organisms, making the precise maintenance of iron homeostasis necessary for survival. While the role of these regulators in iron homeostasis has been well studied in a non-pathogenic E. coli strain, their impact on the production of virulence-associated factors is still unknown in a pathogenic E. coli strain. We thus investigated the role of RyhB and Fur in iron homeostasis and virulence of the uropathogenic E. coli (UPEC) strain CFT073. In a murine model of UTI, deletion of fur alone did not attenuate virulence whereas the ΔryhB and the double Δfur ΔryhB mutants were significantly reduced in bladder colonization. The Δfur mutant was more sensitive to oxidative stress and produced more of the siderophores enterobactin, salmochelins and aerobactin than the wild-type strain. By contrast, while RyhB was not implicated in oxidative stress resistance, the ΔryhB mutant produced less siderophores. This decrease was correlated with the downregulation of genes implicated in siderophore biosynthesis such as shiA (enterobactin and salmochelins) and iucD (aerobactin) in this mutant grown in minimal medium and in human urine. iucD was also downregulated in bladders infected with the ΔryhB mutant compared to the wild-type strain. Our results thus demonstrate that the sRNA RyhB is involved in production of iron acquisition systems and colonization of the urinary tract by pathogenic E. coli.