Ca2+-sensing receptor (CaSR) represents a potential therapeutic target for inflammatory bowel diseases and strongly prefers aromatic amino acid ligands. We investigated the regulatory effects of dietary supplementation with aromatic amino acids – tryptophan, phenylalanine and tyrosine (TPT) – on the CaSR signalling pathway and intestinal inflammatory response. The in vivo study was conducted with weanling piglets using a 2 × 2 factorial arrangement in a randomised complete block design. Piglets were fed a basal diet or a basal diet supplemented with TPT and with or without inflammatory challenge. The in vitro study was performed in porcine intestinal epithelial cell line to investigate the effects of TPT on inflammatory response using NPS-2143 to inhibit CaSR. Dietary supplementation of TPT alleviated histopathological injury and decreased myeloperoxidase activity in intestine challenged with lipopolysaccharide. Dietary supplementation of TPT decreased serum concentration of pro-inflammatory cytokines (IL-1β, IL-6, IL-8, IL-12, granulocyte-macrophage colony-stimulating factor, TNF-α), as well as the mRNA abundances of pro-inflammatory cytokines in intestine but enhanced anti-inflammatory cytokines IL-4 and transforming growth factor-β mRNA levels compared with pigs fed control diet and infected by lipopolysaccharide. Supplementation of TPT increased CaSR and phospholipase Cβ2 protein levels, but decreased inhibitor of NF-κB kinase α/β and inhibitor of NF-κB (IκB) protein levels in the lipopolysaccharide-challenged piglets. When the CaSR signalling pathway was blocked by NPS-2143, supplementation of TPT decreased the CaSR protein level, but enhanced phosphorylated NF-κB and IκB levels in IPEC-J2 cells. To conclude, supplementation of aromatic amino acids alleviated intestinal inflammation as mediated through the CaSR signalling pathway.