Iron availability plays an essential role in staphylococcal pathogenesis. We selected FhuD2, a lipoprotein involved in iron-hydroxamate uptake, as a novel vaccine candidate against Staphylococcus aureus. Unprecedented for staphylococcal lipoproteins, the protein was demonstrated to have a discrete, punctate localization on the bacterial surface. FhuD2 vaccination generated protective immunity against diverse clinical S. aureus isolates in murine infection models. Protection appeared to be associated with functional antibodies that were shown to mediate opsonophagocytosis, to be effective in passive transfer experiments, and to potentially block FhuD2-mediated siderophore uptake. Furthermore, the protein was found to be up-regulated in infected tissues and was required for staphylococcal dissemination and abscess formation. Herein we show that the staphylococcal iron-hydroxamate uptake system is important in invasive infection and functions as an efficacious vaccine target. The extraordinary speed of antibiotic resistance acquisition of Staphylococcus aureus, associated with the severity of staphylococcal infections, dictates the urgent need of a vaccine against this deadly pathogen. However, although several vaccine candidates have been proposed and some of them have been tested in clinical trials using both active and passive immunization modalities [1], an effective vaccine is still missing. In an attempt to develop an S. aureus vaccine, we have been applying a reverse vaccinology approach, mainly based on in silico screening and proteomics, to select surface-exposed proteins. As reported in this work, FhuD2 (ferric hydroxamate uptake) is one of the proteins identified through our screening. FhuD2 was first discovered as a ferric hydroxamate siderophore binding protein that mediates iron uptake through a