Salmonella enterica serovars Typhi and Typhimurium are two closely related bacteria causing different types of infection in humans. Iron acquisition is considered essential for virulence. Siderophores are important iron chelators and production of enterobactin and salmochelins by these serovars was quantified. Overall, Salmonella Typhi produced higher levels of siderophores than Salmonella Typhimurium. The role of the global regulator Fur, involved in iron homeostasis, present and conserved in both these serovars, was then investigated. Deletion of the fur gene led to distinct phenotypes in these serovars. Defective growth in iron-rich and iron-limiting conditions and formation of filamentous cells was only observed in the S. Typhi fur mutant. Furthermore, Fur was required for optimal motility in both serovars, but motility was more reduced for the fur mutant of S. Typhi compared to S. Typhimurium. During interaction with human-cultured macrophages, Fur was more important for S. Typhi, as the fur mutant had severe defects in uptake and survival. Globally, these results demonstrate that Fur differentially affects the physiology and the virulence phenotypes of the two strains and is more critical for S. Typhi growth, morphology, motility and interaction with host cells than it is for S. Typhimurium.