Conclusion: These results indicate that Gln might stimulate the systemic innate immune responses of the spleen in broiler chickens challenged with S. pullorum. Background: The effects of glutamine on the growth performance and systemic innate immune response in broiler chickens challenged with Salmonella pullorum were evaluated. A total of 600 one-day-old Arbor Acres broiler chickens were assigned randomly to 6 dietary treatments with 10 replicates for a 21-day feeding experiment. The experimental treatments were as follows: the control treatment (birds fed the basal diet), the Gln1 treatment, and the Gln 2 treatment (birds fed the basal diet supplemented with 0.5%, and 1.0% Glutamine, respectively). At 3 d of age, birds from each treatment were then oral gavage with 2.0 × 104 CFU/mL of S. pullorum suspension (1.0 mL per bird) or an equivalent amount of sterile saline alone, which served as a control. Results: The results showed that S. pullorum infection had adverse effects on the average daily feed intake, average daily gain, and feed conversion ratio of broiler chickens compared with those of the CON treatment on d 7, decreased the spleen and bursa of Fabricius relative weights (except on d 21), serum immunoglobulin A (IgA),immunoglobulin G (IgG), and immunoglobulin M (IgM) concentrations, and spleen melanoma differentiation-associated gene 5 (MDA5) and laboratory of genetics and physiology gene 2 (LGP2) mRNA expression levels, and increased the mRNA expression levels of spleen Nodinitib-1 (NOD1), Toll-like receptors 2,4 (TLR2, TLR4), DNA-dependent activator of IFN-regulatory factors (DAI), mitochondrial antiviral-signaling protein (MAVS), P50, P65, and RelB on d 4, 7, 14, and 21. Supplementation with Gln improved the relative weights of the spleen and bursa of Fabricius (except on d 21), increased the serum IgA, IgG, and IgM concentrations and the mRNA expression levels of spleen MDA5 and LGP2, and decreased the mRNA expression levels of spleen NOD1, TLR2, TLR4, DAI, MAVS, P50, P65, and RelB of S. pullorum-challenged broiler chickens.