Cysteine and tryptophan are amino acids that have non-nutritional anti-inflammatory and anti-oxidative functions. The aim of this study was to determine the effectiveness of cysteine and tryptophan supplementation on dextran sodium sulphate (DSS)-induced colitis in piglets. DSS was administered via intra-gastric catheter for 5 days at 1.25g·DSS/kg·body weight and cysteine and tryptophan were administered similarly at about 60% and 80% respectively of the recommended daily requirement for 5 days. Growth and histology parameters showed that cysteine and tryptophan treatment groups were able to improve weight to feed conversion ratio and histological parameters when compared with the positive group, which received DSS but no supplementation of amino acids. Similarly, D-mannitol gut permeability assay showed that the amino acid treated groups had reduced gut permeability. Lastly, pro-inflammatory cytokine concentrations and mRNA expression profiles showed improvements in amino acid treated groups. Therefore, cysteine and tryptophan provided therapeutic protection against DSS-induced colitis in piglets.