The present study evaluated whether dietary arginine (Arg) supplementation could attenuate immune challenge induced by Salmonella enterica serovar Choleraesuis C500 (S.C500) through the Toll-like receptor (TLR) 4-myeloid differentiation factor 88 (Myd88) signalling pathway in weaned piglets. A total of thirty-six weaned pigs were randomly allocated into six groups with six replicates per group. Pigs were subjected to three dietary treatments (namely two groups per treatment) in the first week (0–7 d) and fed with diets containing 0, 0·5 and 1·0 % l-Arg, respectively. On day 8, pigs were injected intramuscularly either with S.C500 or sterile saline. Serum samples were collected at day 8 (before injection), and at 1, 3 and 10 d post-injection, pigs were killed for evaluation of tissue gene expression following the last blood collection. Piglets fed the diets with 0·5 or 1·0 % Arg supplementation had a higher concentration of serum Arg (P < 0·05). S.C500-challenged piglets had higher (P < 0·05) serum antibody levels during the days 9–18. Weight gain and feed intake were decreased remarkably (P < 0·01) after the injection of S.C500, and 0·5 or 1·0 % Arg supplementation tended to alleviate the inhibition. The S.C500 challenge significantly enhanced (P < 0·05) serum C-reactive protein (CRP), interferon-γ and IL-12 concentrations, but Arg supplementation attenuated (P < 0·05) the increase in CRP level. The mRNA expression of TLR4, TLR5, Myd88, p65 NF-κB and TNF-α was up-regulated (P < 0·05) by the S.C500 challenge in different tissues, but was down-regulated (P < 0·05) by Arg supplementation. In conclusion, Arg supplementation could inhibit the excessive activation of the TLR4-Myd88 signalling pathway and thus attenuated the negative effects caused by the immune challenge of S.C500.