Dysbiosis of intestinal microbiota may cause irregular digestive function, and intestinal wall inflammation. Over the past few years, probiotics generate bioactive metabolites, named postbiotics, have been discovered its crucial roles in modulation of intestinal microbiota. Single-strain postbiotics have positive effect on health of host, but the functions of multi-strain postbiotics remain unclear. This study proposed a useful application of multi-strain postbiotics and thereby establish the developing foundation of multi-strain postbiotics. Initially, various probiotics and postbiotics were screened for anti-inflammatory activity through inducing the transforming growth factor beta (TGF-β)and interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMCs). Then, we detailed the synergistic effects of 4Mix postbiotics (named as Probiotic Extracts of 4 strains- number 1, PE0401) consisted of metabolites generated from Lactobacillus salivarius AP-32, Lactobacillus acidophilus TYCA06, Lactobacillus plantarum LPL28, Bifidobacterium longum subsp. infantis BLI-02 on anti-inflammatory activity, anti-oxidative capacity, regulation of tight junction proteins. The results displayed that anti-inflammatory activity of 4Mix postbiotics PE0401 was stronger than other mixed postbiotic combinations. The anti-oxidative capacity, which correlated to anti-inflammation, also significantly increased as shown in DPPH and FRAP assays. The epithelial tight junction proteins expressed in mRNA levels (ZO-1, ZO-2, Occludin, JAM-A, and Claudin) were highly potent modulated by PE0401. In addition, PE0401 selectively promoted the growth of intestinal bacterial strains including Lactobacillus, Bifidobacterium strains and other beneficial bacteria. Therefore, this study provides a fascinating insight into the strategy to the treatment of the intestinal disorders. PE0401 may deliver as health functional food ingredient.