The spirochetes of the Leptospira genus contain saprophytic and pathogenic members, the latter being responsible for leptospirosis. Despite the recent sequencing of the genome of the pathogen L. interrogans, the slow growth of these bacteria, their virulence in humans, and a lack of genetic tools make it difficult to work with these pathogens. In contrast, the development of numerous genetic tools for the saprophyte L. biflexa enables its use as a model bacterium. Leptospira spp. require iron for growth. In this work, we show that Leptospira spp. can acquire iron from different sources, including siderophores. A comparative genome analysis of iron uptake systems and their regulation in the saprophyte L. biflexa and the pathogen L. interrogans is presented in this study. Our data indicated that, for instance, L. biflexa and L. interrogans contain 8 and 12 genes, respectively, whose products share homology with proteins that have been shown to be TonB-dependent receptors. We show that some genes involved in iron uptake were differentially expressed in response to iron. In addition, we were able to disrupt several putative genes involved in iron acquisition systems or iron regulation in L. biflexa. Comparative genomics, in combination with gene inactivation, gives us significant functional information on iron homeostasis in Leptospira spp.