This study was conducted to examine the effects of fructooligosaccharide (FOS) supplementation on growth performance, lymphoid organ weight, intestinal morphology, and immunological status in broilers (n=180) challenged with Salmonella Enteritidis lipopolysaccharides (LPS). Birds were randomly assigned into a 3×2 factorial arrangement that included 1) 3 dietary treatments from d one to 21: positive control (PC), wheat-corn-soybean meal based diet contained antibiotics (virginiamycin and monensin); negative control (NC), as PC without antibiotics; and NC+FOS, as NC supplemented with 0.5% FOS, and 2) 2 intraperitoneal injections: 2 mg/kg Salmonella Enteritidis LPS or sterile phosphate buffered saline (PBS) on d 21. Growth performance and relative lymphoid organ weight were not significantly different among the treatments. Villus height, crypt depth, and total mucosa thickness were significantly increased (P<0.05) in the ileum of broiler chickens fed NC+FOS when compared to PC and NC. Birds in NC+FOS treatment had reduced heterophil but increased monocyte count when compared to NC (P<0.05). Significant diet×challenge interaction was observed on natural IgY levels (P<0.0001), and a significant dietary effect was observed on specific IgY levels in chickens fed NC+FOS (P=0.003). Supplementation of FOS also increased the expression of interleukin (IL)-1ß, -10, and interferon (IFN)-γ mRNA in the ileum of the birds. In summary, Salmonella Enteritidis LPS challenge established significant differences in the immune responses in broiler chickens. FOS supplementation increased ileal mucosa thickness and elevated the expressions of certain cytokine genes. It also led to the alteration of leukocyte compositions and serum IgY levels in response to LPS challenge, suggesting FOS supplementation may be effective to induce protective outcomes in gut health and immunity of broiler chickens.