Flagellin is an important stimulus for epithelial interleukin-8 (IL-8) secretion because of its ability to activate Toll-like receptor 5 (TLR5). SopE2, a Salmonella guanine nucleotide exchange factor (GEF), is also involved in intestinal inflammation. To clarify the proinflammatory mechanisms of these proteins, we examined their effects on IL-8 secretion and intracellular signaling in T84 epithelial cells. A Salmonella strain lacking SopE2 (and its homolog SopE) induced lower levels of IL-8 than the wild type and exhibited reduced activation of mitogen-activated protein kinases (MAPKs). Overexpression of wild-type SopE2 in this strain restored MAPK activation and augmented IL-8 production, whereas a mutant lacking GEF activity failed to increase IL-8 expression. Additional effects on signaling were demonstrated in transient transfection experiments, in which SopE2 enhanced the ability of TRAF6, a signal transducer downstream of TLR5, to activate the NF-kappaB transcription factor in 293 cells. Flagellin was also found to be required for IL-8 induction in T84 cells. In its absence, the ability of SopE2 overexpression to increase IL-8 secretion was impaired. Part of this impairment was related to the decreased motility of the flagellin-deficient strain, but lack of flagellin also affected translocation of SopE2 into the infected cells. Our results indicate that flagellin and SopE2 interact functionally at multiple levels to increase IL-8 secretion by epithelial cells-flagellin facilitating the translocation of SopE2, and SopE2 enhancing signaling pathways activated by flagellin. These observations offer a mechanistic explanation for the involvement of these proteins in the pathogenesis of Salmonella-induced gastroenteritis.