Flagellin is a bacterial protein responsible for activation of toll-like receptor 5 (TLR5) which we hypothesize is involved in Salmonella's induction of cytoprotective heat shock proteins in intestinal epithelial cells. Flagellin induces the cytoprotective heat shock protein Hsp25 in different intestinal epithelial cell lines and in mouse intestine. Flagellin induces Hsp25 expression in a time-dependent manner in vitro. This effect is transcriptional as confirmed by luciferase reporter assays and actinomycin D treatment. In addition, Hsp25 induction requires p38 MAP kinase activation and is only observed when flagellin is added to the basolateral side of polarized intestinal epithelial cells, consistent with the known location of TLR5. Flagellin-mediated Hsp25 induction is associated with increased protective effects against oxidant stress, an effect that is at least partially mediated by p38 MAP kinase. Use of siRNA against Hsp25 demonstrates that flagellin-mediated protection against oxidant stress is to some degree mediated through Hsp25 induction. This suggests that, by protecting against oxidant injury, the induction of Hsp25 expression by flagellin may contribute to intestinal homeostasis. In a co-culture cell model and in a mouse model of Salmonella enterica serovar Typhimurium infection, not only does infection with wild-type and a flagellin-deletion mutant strain of Salmonella show that flagellin induces Hsp25 in vivo but it also demonstrates that in the case of live Salmonella infection, flagellin serves as a major stimulus for the induction of Hsp25 expression. These data provide evidence that flagellin is required for Salmonella-mediated induction of Hsp25 expression in intestinal epithelium.