Type III secretion systems (TTSS) are used by Gram-negative pathogens to translocate proteins into eukaryotic host cells. Salmonella enterica serovar Typhimurium (S. Typhimurium) has two of these specialized systems, which are encoded on separate Salmonella pathogenicity islands (SPI-1 and SPI-2) and translocate unique sets of effectors. The specific roles of these systems in Salmonella pathogenesis remain undefined, although SPI-1 is required for bacterial invasion of epithelial cells and SPI-2 for survival/replication in phagocytic cells. However, because SPI-1 TTSS mutants are invasion-incompetent, the role of this TTSS in post-invasion processes has not been investigated. In this study, we have used two distinct methods to internalize a non-invasive SPI-1 TTSS mutant (invA) into cultured epithelial cells: (i) co-internalization with wild-type S. Typhimurium (SPI-1-dependent) and (ii) complementation with the Yersinia pseudotuberculosis invasin (inv) gene (SPI-1-independent). In both cases, internalized invA mutants were unable to replicate intracellularly, indicating that SPI-1 effectors are essential for this process and cannot be complemented by wild-type bacteria in the same cell. Analysis of the biogenesis of SCVs showed that vacuoles containing mutant bacteria displayed abnormal maturation that was dependent on the mechanism of entry. Manipulation of Salmonella-containing vacuole (SCV) biogenesis by pharmacologically perturbing membrane trafficking in the host cell increased intracellular replication of wild-type but not mutant S. Typhimurium This demonstrates a previously unknown role for SPI-1 in vacuole biogenesis and intracellular survival in non-phagocytic cells.