Bacterial pathogens require multiple iron-specific acquisition systems for survival within the iron-limiting environment of the host. Francisella tularensis is a virulent intracellular pathogen that can replicate in multiple cell-types. To study the interrelationship of iron acquisition capability and virulence potential of this organism, we generated single and double deletion mutants within the ferrous iron (feo) and ferric-siderophore (fsl) uptake systems of the live vaccine strain (LVS). The Feo system was disrupted by a partial deletion of the feoB gene (ΔfeoB'), which led to a growth defect on iron-limited modified Muller Hinton agar plates. 55Fe uptake assays verified that the ΔfeoB' mutant had lost the capacity for ferrous iron uptake but was still competent for 55Fe-siderophore-mediated ferric iron acquisition. Neither the ΔfeoB' nor the siderophore-deficient ΔfslA mutant was defective for replication within J774A.1 murine macrophage-like cells, thus demonstrating the ability of LVS to survive using either ferrous or ferric sources of intracellular iron. A LVS ΔfslA ΔfeoB' mutant defective for both ferrous iron uptake and siderophore production was isolated in the presence of exogenous F. tularensis siderophore. In contrast to the single deletion mutants, the ΔfslA ΔfeoB' mutant was unable to replicate within J774A.1 cells and was attenuated in virulence following intraperitoneal infection of C57BL/6 mice. These studies demonstrate that the siderophore and feoB-mediated ferrous uptake systems are the only significant iron acquisition systems in LVS and that they operate independently. While one system can compensate for loss of the other, both are required for optimal growth and virulence.