The Disulfide Bond Formation (Dsb) System is important for maintaining the structure and function of periplasmic proteins. Although the biochemistry of various Dsb proteins has been characterized in E. coli, the potential link between this system and virulence mechanisms of pathogenic Gram-negative bacteria has not been elucidated. Deletion mutations generated in dsb genes demonstrated multiple virulence defects in Salmonella enterica serovar Enteritidis, which were in turn rescued by complementation. Analyses revealed that two members of the Dsb system, DsbA and DsbC, were required for invasion of epithelial cells, a process mediated by the Type III Secretion System (T3SS) encoded by Salmonella Pathogenicity Island 1 (SPI-1). The Dsb system was also required for colonization and virulence in vivo. Strains expressing T3SS effectors tagged with a FLAG epitope were transduced with the dsb mutations, and analyses of the supernatant fractions indicated that the Dsb system was involved in the secretion of SipA and SipC, which are critical for ruffling of the host cell membrane and subsequent internalization of the bacteria. A bacterial two-hybrid assay was performed to identify Dsb substrates among the T3SS needle complex proteins, and preliminary results indicated direct binding of the Dsb system with SpaO, a T3SS sorting platform protein. These results suggested that the formation and isomerization of disulfide bonds are critical steps in the function of the T3SS which contributes to the pathogenesis of Salmonella.