Chronic stress is associated with detrimental effects on physical health, such as chronic restraint stress (CRS), which can damage the intestinal tract. Although tryptophan has many benefits in maintaining intestinal health, the underlying mechanism of its protective effects against stress-induced intestinal injury remains unclear. In this study, we constructed a CRS model by using a behavioral restraint device in which mice were restrained for 6 h per day over 14 days and investigated the effects, as well as the potential mechanism of a high-tryptophan diet (0.4% tryptophan), on CRS-induced intestinal injury using scanning electron microscopy, 16S rRNA sequencing, and LC-MS. A 0.4% tryptophan diet (fed ad libitum for 24 days) attenuated CRS-induced pathologies, including weight loss, elevated corticosterone, intestinal barrier injury, increased permeability, and epithelial apoptosis. Tryptophan modulated the gut microbiota composition in CRS-induced mice, increasing the abundance of and decreasing the abundance of , as well as enhancing metabolic function through pathways identified by KEGG analysis. Additionally, tryptophan restored the levels of short-chain fatty acids (SCFAs), including acetic, propionic, isobutyric, butyric, and valeric acids. Correlation analyses showed interactions between tryptophan, intestinal permeability, SCFAs, and gut microbiota. Tryptophan supplementation attenuates CRS-induced intestinal injury by modulating intestinal barrier integrity and gut microbiota homeostasis, and the beneficial effects are largely associated with the SCFA-mediated regulation of intestinal permeability and microbiota-associated energy metabolism.