Competition for cellular iron (Fe) is a vital component of the interaction between host and intracellular pathogen. The host cell requires Fe for the execution of antimicrobial effector mechanisms, whereas most bacteria have an obligate requirement for Fe to sustain growth and intracellular survival. In this study, we show that chelation of host Fe in vivo exacerbates murine salmonellosis, resulting in increased bacterial load and decreased survival times. We further demonstrate that host Fe deprivation results in an inability to induce the NADPH oxidase-dependent production of reactive oxygen, an essential host defense mechanism for the early control of Salmonella typhimurium infection. Thus, altering the equilibrium of intracellular Fe influences the course of infection to the benefit of the pathogen.