Salmonella pathogenicity island 2 (SPI2) encodes a type III secretion system (TTSS) necessary for bacterial survival and replication in intracellular environment of host cells. SPI2 genes are transcribed preferentially after Salmonella enters the host cells. Transcriptional regulation of ssaG encoding the component of SPI2-TTSS apparatus was studied in vivo and in vitro. Fis, one of the major components of bacterial nucleoid, activated the stationary phase-specific expression of ssaG when Salmonella was grown in LB media. Gel-shift and footprinting analysis showed Fis bound to four distinct sites of the ssaG promoter region with different affinities. All four Fis-binding sites were required for timely transcription activation of ssaG after Salmonella entered macrophage cells. Gentamicin protection experiments using bacteria grown to stationary phase prior to infection showed that the ability of the fis mutant strain to replicate within the RAW264.7 macrophage cells was lower than the wild type. These observations confirm that Fis plays an important role in regulations of SPI2 as well as SPI1 for an efficient regulation of the virulence genes.