Iron is an important nutritional requirement for bacteria due to its conserved role in many essential metabolic processes. As a consequence of the lack of freely available iron in the mammalian host, bacteria upregulate a range of virulence factors during infection. Transcriptional analysis of Francisella tularensis subsp. novicida U112 grown in iron-deficient medium identified 21 genes upregulated in response to this condition, four of which were attributed to a siderophore operon. In addition, a novel iron-regulated gene, FTT0025, was identified which is part of this operon and encodes a 55 kDa hypothetical membrane protein. When grown on chrome azurol S agar, the F. tularensis subsp. novicida U112deltaFTT0025 mutant produced an increased reaction zone compared with the wild-type, suggesting that siderophore production was unaffected but that the bacteria may have a deficiency in their ability to re-sequester this iron-binding molecule. Furthermore, the deltaFTT0025 mutant was attenuated in a BALB/c mouse model of infection relative to wild-type F. tularensis subsp. novicida U112.