Ulcerative colitis (UC) is closely related to impaired intestinal barrier function and imbalanced gut microbial communities. l-theanine shows great potential in maintaining intestinal integrity and regulating the gut microbiota and associated short-chain fatty acids (SCFAs). However, whether l-theanine can alleviate UC by repairing the intestinal barrier through these regulatory processes remains unclear. In this study, l-theanine was used to optimize the gut microbiota, and the restorative effect and mechanism of l-theanine in UC by repairing the gut barrier through the gut microbiota and SCFAs were investigated via fecal microbiota transplantation. The findings revealed that l-theanine regulated the gut microbiota structure, increased SCFA contents, and promoted gut barrier repair in UC mice. Moreover, l-theanine upregulated the protein and mRNA expression of G-protein-coupled receptor 43 (GPR43), AKT, and phosphatidylinositide 3-kinase (PI3K). These results indicated that l-theanine alleviates UC by repairing the gut barrier via regulating the gut microbiota and SCFAs through the GPR43/PI3K/AKT signaling pathway activation. This study provides a method of preventing and treating UC via l-theanine as a safe food dietary supplement.