Gliadin, abundant in flour-based foods and processed foods, has been widely researched for allergies. However, the impact of gliadin on the intestinal barrier of healthy individuals and the intervention effect of Bifidobacterium longum (B. longum) are rarely explored. Three strains (JCM1217, CCFM1216, CCFM1218) of B. longum with strong gliadin hydrolysis were screened from 18 strains. This study explored the effects of B. longum on mice with a 10-week high-fat diet and 6% gliadin (HFD + 6%G), assessing duodenal health, lipid metabolism, metabolomics, and gut microbiota in the duodenum and colon changes. Three B. longum strains were screened for gliadin hydrolysis to produce minimal R5 immunopeptide production. All three B. longum strains improved duodenal morphology, reduced intestinal permeability, reduced inflammation (IL-15), and activated tryptophan metabolism. Additionally, alterations in the microbiota of the duodenum and colon were also observed. Linear discriminant analysis (LDA) showed that the HFD + 6% G group significantly increased the abundance of Ileibacterium, Alistipes, Bacteroides, Candidatus, Saccharimonas, Streptococcus, Sediminibacterium, and Odoribacterium in the duodenum. The abundance of Blautia, Butyricimonas, Ruminococcaceae UCG-010, Parabacterioids, and Eubacterium nodatum in the colon was also increased. The B. longum CCFM1216 and B. longum CCFM1218 reversed the abundance of these strains. Specifically, B. longum CCFM1216 enhanced the duodenal barrier with indoleacrylic acid, beneficial for blood lipids and glucose. These strains may be used as probiotics for gliadin-related diseases.