In the present study, two experiments were conducted to investigate the effect of dietary L-arginine (Arg) supplementation on the inflammatory response and innate immunity of broiler chickens. Expt 1 was designed as a 2 × 3 factorial arrangement (n 8 cages/treatment; n 6 birds/cage) with three dietary Arg concentrations (1.05, 1.42 and 1.90%) and two immune treatments (injection of lipopolysaccharide (LPS) or saline) given at an interval of 48 h between 14 and 21 d of age. In Expt 2, correlation between dietary Arg concentration (0.99, 1.39, 1.76, 2.13 or 2.53%) and percentage of circulating B cells (percentage of circulating lymphocytes) was determined. In Expt 1, LPS injection decreased body-weight gain and feed intake and increased feed conversion ratio of the challenged broilers (14-21 d; P< 0.05). LPS injection suppressed (P< 0.05) the percentages of splenic CD11+ and B cells (percentages of splenic lymphocytes) and phagocytic activity of splenic heterophils and macrophages; Arg supplementation linearly decreased the percentages of CD11+, CD14+ and B cells in the spleen (P< 0.10). LPS injection increased (P< 0.05) the expression of IL-1β and IL-6 mRNA in the spleen and caecal tonsils. Arginine supplementation decreased (P< 0.05) the expression of IL-1β, Toll-like receptor 4 (TLR4) and PPAR-γ mRNA in the spleen and IL-1β, IL-10, TLR4 and NF-κB mRNA in the caecal tonsils. In Expt 2, increasing dietary Arg concentrations linearly and quadratically reduced the percentage of circulating B cells (P< 0.01). Collectively, Arg supplementation attenuated the overexpression of pro-inflammatory cytokines probably through the suppression of the TLR4 pathway and CD14+ cell percentage. Furthermore, excessive Arg supplementation (1.76%) suppressed the percentages of circulating and splenic B cells.