Salmonella Typhimurium (ST) infection in chickens inhibits their growth and can lead to food-borne diseases in humans. Probiotics are expected to enhance the function of host intestinal barrier against pathogen infection. The aim of our study was to determine the effect of viable Lactobacillus reuteri (LR) on the response of the mucosal barrier function to antigen stimulation in broiler chicks. Day-old male (n＝8) and female (n＝4) broiler chicks were orally administered either 1×10 8 LR or a water-only control, every day for 7 days. After 7 days, either 1×10 8 heatkilled ST (k-ST), or a buffer-only control, was administered via intra-cardiac injection. The ileum and cecum were collected 3 h post-injection, and paraffin sections were prepared for either mRNA extraction (males), or gut permeability tests (females). Villus and crypt lengths were determined via histological analysis. Real-time PCR was used to calculate expression levels of Toll-like receptors (TLRs), pro-inflammatory cytokines, anti-inflammatory cytokines, avian β-defensins, and tight-junction-associated molecules. Gut permeability was assessed using the inverted intestine method. We found that (1) expression of TLR2-1, TLR21, TGF-β2 and TGF-β3 were reduced following k-ST stimulation, but were unaffected by LR-treatment; (2) oral administration of LR led to increased Claudin1, Claudin5, ZO2, and JAM2 expression following k-ST stimulation; (3) cecal permeability was reduced by co-treatment with LR and k-ST, but not by treatment with LR or k-ST alone. These results suggest that LR, as used in this study, may enhance the intestinal mucosal physical barrier function, but not the expression of other immunerelated factors in newly hatched chicks.