Heat shock proteins (Hsp), Hsp25 and Hsp72 are expressed normally by surface colonocytes, but not by small intestinal enterocytes. We hypothesized that luminal commensal microflora, maintain the observed colonocyte Hsp expression. The ability of the small intestine to respond to bacteria and their products and modulate hsps has not been determined. The effects of luminal bacterial flora in surgically-created mid-jejunal self-filling (SFL) versus self-emptying (SEL) small bowel blind loops on epithelial Hsp expression were studied. Hsp25 and Hsp72 expression were assessed by immunoblot and immunohistochemistry. SFL were chronically colonized, whereas SEL contained levels of bacteria normal for the proximal small intestine. SFL creation significantly increased Hsp 25 and 72 expression relative to corresponding sections from SEL. Metronidazole treatment, which primarily affects anaerobic bacteria, as well as a diet lacking fermentable fiber, significantly decreased SFL Hsp expression. Small bowel incubation with butyrate ex vivo induced a sustained and significant up-regulation of Hsp25 and altered Hsp72 expression, confirming the role of short chain fatty acids. To determine if Hsps induction altered responses to an injury, effects of the oxidant, monochloramine, on epithelial resistance (R) and short circuit (Isc) responses to carbachol and glucose were compared. Increased SFL Hsp expression was associated with protection against oxidant-induced decreases in transmural resistance and short-circuit responses to glucose, but not secretory responses to carbachol. In conclusion, luminal microflora and their metabolic byproducts direct expression of Hsps in gut epithelial cells, an effect that contributes to preservation of epithelial cell viability under conditions of stress.