Salmonella invade non-phagocytic cells by inducing massive actin rearrangements, resulting in membrane ruffle formation and phagocytosis of the bacteria. This process is mediated by a cohort of effector proteins translocated into the host cell by type III secretion system 1, which is encoded by genes in the Salmonella pathogenicity island (SPI) 1 regulon. This network is precisely regulated and must be induced outside of host cells. In vitro invasive Salmonella are prepared by growth in synthetic media although the details vary. Here, we show that culture conditions affect the frequency, and therefore invasion efficiency, of SPI1-induced bacteria and also can affect the ability of Salmonella to adapt to its intracellular niche following invasion. Aerobically grown late-exponential-phase bacteria were more invasive and this was associated with a greater frequency of SPI1-induced, motile bacteria, as revealed by single-cell analysis of gene expression. Culture conditions also affected the ability of Salmonella to adapt to the intracellular environment, since they caused marked differences in intracellular replication. These findings show that induction of SPI1 under different pre-invasion growth conditions can affect the ability of Salmonella to interact with eukaryotic host cells.