Citric acid promotes immune function by modulating the intestinal barrier. This study demonstrates that citric acid enhances intestinal epithelial cell growth, increases the villus-crypt ratio, and promotes the growth of beneficial gut bacteria such as Bifidobacterium and Lactobacillus. It also increases the expression of tight junction proteins like occludin, ZO-1, and claudin-1, which are crucial for maintaining the intestinal barrier. In H9N2 influenza virus-infected IEC-6 cells, citric acid enhances the expression of genes related to viral infection, reduces inflammatory responses, and inhibits viral replication. These findings suggest that citric acid strengthens the intestinal barrier, inhibits influenza virus replication, and boosts intestinal immune function. The study used C57BL/6 mice and IEC-6 cells to investigate the molecular effects of citric acid on the intestinal barrier, employing techniques such as RT-PCR, RT-qPCR, flow cytometry, and metabolomics analysis. The results indicate that citric acid promotes cell proliferation, improves intestinal structure, and enhances the expression of tight junction genes. Additionally, citric acid modulates serum metabolism, affecting pathways such as taurine, arachidonic acid, and amino acid metabolism. The study also shows that citric acid reduces the expression of inflammatory factors like TNF-α and IL-6 in H9N2-infected cells, thereby mitigating the inflammatory response and inhibiting viral replication. Overall, citric acid enhances intestinal barrier function, supports immune health, and may serve as a potential alternative to antibiotics in promoting gut health.Citric acid promotes immune function by modulating the intestinal barrier. This study demonstrates that citric acid enhances intestinal epithelial cell growth, increases the villus-crypt ratio, and promotes the growth of beneficial gut bacteria such as Bifidobacterium and Lactobacillus. It also increases the expression of tight junction proteins like occludin, ZO-1, and claudin-1, which are crucial for maintaining the intestinal barrier. In H9N2 influenza virus-infected IEC-6 cells, citric acid enhances the expression of genes related to viral infection, reduces inflammatory responses, and inhibits viral replication. These findings suggest that citric acid strengthens the intestinal barrier, inhibits influenza virus replication, and boosts intestinal immune function. The study used C57BL/6 mice and IEC-6 cells to investigate the molecular effects of citric acid on the intestinal barrier, employing techniques such as RT-PCR, RT-qPCR, flow cytometry, and metabolomics analysis. The results indicate that citric acid promotes cell proliferation, improves intestinal structure, and enhances the expression of tight junction genes. Additionally, citric acid modulates serum metabolism, affecting pathways such as taurine, arachidonic acid, and amino acid metabolism. The study also shows that citric acid reduces the expression of inflammatory factors like TNF-α and IL-6 in H9N2-infected cells, thereby mitigating the inflammatory response and inhibiting viral replication. Overall, citric acid enhances intestinal barrier function, supports immune health, and may serve as a potential alternative to antibiotics in promoting gut health.