To investigate the feature of amino acids metabolism of human colonic microbes and the influence of their metabolites on the permeability of epithelial cells， in vitro batch fermentation of human gut microbiota was used. The media were based on general 20 amino acids with bovine serum albumin and NaCl as controls. The feces specimen was collected from 27 healthy people aged 20 to 55 years old. The short-chain fatty acids （SCFA） in the broths of 24 h fermentation were detected by gas chromatography. The effects of the amino acids-derived SCFA on the transmembrane resistance of Caco-2 cells was determined. The production of isovaleric acid in the medium containing leucine（Leu） and isoleucine （Ile） was significantly higher than that of the control group （P<0.01）. After fermentation of the medium containing valine （Val）， the level of isobutyric acid was significantly increased （P<0.01）. In the medium containing proline （Pro）， the production of valeric acid was significantly increased （P<0.01）. The results of the cell line showed that valeric acid can significantly increase the transmembrane resistance of Caco-2 cells （P<0.01）， while isobutyric acid and isovaleric acid can significantly reduce the transmembrane resistance. As a conclusion， isovaleric acid in the colon content is formed by the degradation of Leu and Ile； isobutyric acid is formed by the metabolism of Val； valeric acid is mainly formed by the metabolism of Pro. Valeric acid can significantly enhance the intestinal mucosal barrier function.