Macrophages are an important intracellular niche for Salmonella particularly for systemic infection. The interaction of Salmonella with these cells is mediated by two type III secretion systems (TTSS), encoded on Salmonella pathogenicity islands 1 and 2 (SPI1, SPI2), which mediate distinct phases of the pathogen-host cell interaction. The SPI1 TTSS mediates invasion whereas the SPI2 TTSS is required for intramacrophage survival. Importantly, however, Salmonella can enter macrophages by either SPI1-dependent invasion or host cell-mediated phagocytosis. Here, we investigated how the mechanism of internalization affects the intracellular environment and TTSS gene expression. Intracellular bacterial survival depended on the method of entry, because complement-opsonized and SPI1-induced Salmonella initiated replication within 8 h whereas immunoglobulin G (IgG)-opsonized and non-opsonized Salmonella were initially killed. Analysis of vacuolar pH showed that acidification of the Salmonella-containing vacuole occurred more rapidly for non-opsonized or SPI1-induced Salmonella compared with IgG-opsonized or complement-opsonized Salmonella. Finally, quantitative polymerase chain reaction was used to compare the transcriptional profiles of selected SPI1 and SPI2 regulon genes. We found that the magnitude of SPI2 gene induction depended on the mechanism of internalization. Unexpectedly, SPI1 genes, which are rapidly downregulated following SPI1-mediated invasion, were induced intracellularly following phagocytic uptake. These results reveal another level of complexity in pathogen-macrophage interactions.