In order to survive inside macrophages, Salmonella produces a series of proteins encoded by genes within Salmonella pathogenicity island 2 (SPI-2). In the present study, we report that Fur, a central regulator of iron utilization, negatively controls the expression of SPI-2 genes. Time course analysis of SPI-2 expression after the entry of Salmonella into macrophages revealed that SPI-2 genes are induced earlier and at higher levels in the absence of the Fur regulator. It was hypothesized that Fur repressed the SPI-2 expression that was activated during acidification of the phagosome. Indeed, as pH was lowered from pH 7.0 to pH 5.5, the lack of Fur enabled SPI-2 gene expression to be induced at higher pH and to be expressed at higher levels. Fur controlled SPI-2 genes via repression of the SsrB response regulator, a primary activator of SPI-2 expression. Fur repressed ssrB expression both inside macrophages and under acidic conditions, which we ascribe to the direct binding of Fur to the ssrB promoter. Our study suggests that Salmonella could employ iron inside the phagosome to precisely control the timing and levels of SPI-2 expression inside macrophages.