Iron acquisition in Yersinia pestis is fundamental to the success of plague pathogenesis. We have previously identified an ≈5.6 kb region (yfe) of Y. pestis genomic DNA, capable of restoring iron-deficient growth but not siderophore production to an Escherichia coli mutant (SAB11) incapable of synthesizing the siderophore, enterobactin. The yfe locus of Y. pestis, found in both pigmented (Pgm+) and nonpigmented (Pgm−) strains, comprises five genes arranged in two distinct operons (yfeA-D and yfeE). The larger of these, yfeABCD, encodes an ABC transport system, whose expression is iron and Fur regulated and is repressed in cells grown in the presence of manganese. Cells from a Pgm−, Yfe− (ΔyfeAB) mutant strain of Y. pestis exhibited reduced transport of both 55Fe and 54Mn. Furthermore, cells containing an intact yfe locus showed reduced 55Fe uptake when competing amounts of MnCl2 or ZnCl2 were present, whereas 54Mn uptake was inhibited by FeCl3 but not by ZnCl2. Similarly, yfe mutants of Y. pestis exhibited growth defects on media supplemented with the iron chelators 2,2′-dipyridyl or conalbumin. These growth defects were not relieved by supplementation with MnCl2. A ybt−, ΔyfeAB mutant of Y. pestis was completely avirulent in mice infected intravenously (LD50 > 1.7 × 107 cfu) compared with its parental ybt−, yfe+ strain, which had an LD50 of < 12. In addition, compared with its ybt+, yfe+ parent, a ybt+, ΔyfeAB mutant of Y. pestis had an ≈100-fold increase in the LD50 from a subcutaneous route of infection. These data suggest that the Yfe and Ybt systems may function effectively to accumulate iron during different stages of the infectious process of bubonic plague.