Dietary flavour compounds, γ-glutamyl dipeptides, have been shown to activate the calcium sensing receptor (CaSR) via allosteric ligand binding, but their role in modulating intestinal inflammation is unknown. Here we show that activation of CaSR by γ-glutamyl cysteine (γ-EC) and γ-glutamyl valine (γ-EV) can prevent TNF-α-induced inflammatory responses in intestinal epithelial cells (IECs); and explore the underlying mechanism at a cellular level. Human intestinal epithelial cell lines, Caco-2 and HT-29, were used as in vitro model to study intestinal inflammation. The TNF-α-induced secretion of interleukin (IL)-8 from IECs which is recognized as a biomarker indicating the inflammation was reduced by γ-EC and γ-EV. The gene expressions of pro-inflammatory cytokines, including TNF-α, IL-6 and IL-1β, were also inhibited by γ-EC and γ-EV supplementation, which is resulted from the activation of exogenous TNF-α-induced activations of NF-κB and MAPK pathways. The inhibitory effects of γ-EC on pro-inflammatory signaling cascades were abolished by specific anti-CaSR antibody and antagonist (NPS2143) in Caco-2 cells. Our current results demonstrated the anti-inflammatory activity of γ-glutamyl dipeptides in vitro in IECs, and indicated that the activity was primarily via CaSR-mediated activation. For further study of the mechanism underlying CaSR activation interfering with TNF-α-induced inflammation, we identified anti-inflammatory effects of the peptides that were abrogated in β-arrestin2 knockdown IECs. As well, involvement of β-arrestin2 was found to inhibit the TNF-α-dependent pro-inflammatory signalling cascade via cross-talk with the TNF-α receptor (TNFR). Activation of CaSR has potential benefits for ameliorating an impaired gut system. The current study identifies the anti-inflammatory activities of γ-glutamyl peptides including γ-EC and γ-EV in a mouse model of colitis. The supplementation of γ-EC and γ-EV in DSS-induced mice were shown to prevent DSS caused loss of weight, colitic symptoms, and shortening of colon length. The study of colonic morphology also suggested a protective effect of γ-EC and γ-EV supplementation on the lesion developed in DSS colitis mouse colon. The productions of pro-inflammatory cytokines in DSS-treated mice were reduced by γ-EC and γ-EV, while the production of anti-inflammatory cytokine IL-10 was augmented. We also identified the inhibitory effect of γ-EC and γ-EV on pro-inflammatory cytokines secretions being abrogated by pre-treated mice with NPS2143. In conclusion, γ-EC and γ-EV can effectively attenuate intestinal inflammation and restore gut homeostasis via ligand activation of CaSR, and may be a novel therapy for chronic inflammatory conditions such as inflammatory bowel disease (IBD). In order to reveal the signaling pathways regulated by γ-glutamyl dipeptides supplementation, we studied the genomic alterations in a γ-EC-treated DSS colitis mouse model via a microarray study. Total of 481 genes from DSS-induced colonic tissue were regulated by γ-EC supplementation. As well known, the inflammatory signaling events were generally up-regulated by DSS-induced colitis. Accordance with the results of biological function and pathway analyses in current study, expressions of inflammatory mediators including myeloperoxidase, transcriptional factors, and chemotatic cytokines in DSS colitis mouse model were majorly down-regulated by γ-EC. The network analysis also showed γ-EC involved in suppressing signaling events associated with intestinal inflammation and preventing abnormal cellular proliferation. The above genomic profile elucidates the potential mechanism underlying γ-EC ameliorating inflammation and preventing tissue damage in the gut system.