Abstract: The aim of this study was to investigate the effects of co-culture supernatant of Lactobacillus casei(LC)and Bacillus subtiliis natto(BN)on intestinal micro-ecology, mucosal barrier function and immune function in mice with antibiotic-associated diarrhea(AAD). The AAD mouse model was established and the normal saline, LC, BN and co-culture supernatants were administered, respectively, for 4 days. The general conditions of the mice during the intervention were observed. The thymus and spleen weight ratios of different intervention mice were compared. The histopathological changes of the proximal colon lesions were observed. The intestinal microecology, mucosal barrier function and immune function of each group were detected. The results showed that the mice in the model group showed poor mental state, decreased feeding intake and abnormal stool characteristics, which were aggravated with the prolongation of time. After intervention, the above-mentioned states of mice in each group were improved, with the best recovery for the co-culture group. Histopathological results showed that the intestinal wall of the model group was severely damaged and villus was shedding. Cellulose-like exudation, necrosis of epithelial cells and infiltration of inflammatory cells could be seen in the model group. The pathological changes mentioned above were improved after intervention, and the co-culture group had the best effect. Compared with the control group, the thymus and spleen weight ratio, microbial diversity(Shannon)index, richness(Chao)index, Lactobacillus number, Bifidobacterium number, secretory immunoglobulin IgA(sIgA)in intestinal mucosa, interleukin(IL)-2 and IL-2/IL-4, the relative expressions of tight junction related protein-1(ZO-1)and atresia protein(Occludin)in intestinal tissue of the model group were lower, while the number of enterobacteria, enterococcus number, serum diamine oxidase(DAO)bacterial ectopic rate and IL-4 in intestinal tissue were higher(P< 0. 05). Compared with the model group, the thymus, spleen weight ratio, Shannon, Chao index, Lactobacillus number, Bifidobacterium number, sIgA in intestinal mucosa, IL-2 and IL-2/IL-4, the relative expressions of ZO-1 and Occludin in intestinal tissue of the intervention groups were higher(P< 0. 05), and the co-culture group was higher than the LC group and the BN group(P< 0. 05). There was no significant difference between the LC group and the BN group(P> 0. 05). Compared with the model group, the number of enterobacteria, enterococcus, serum DAO, bacterial ectopic rate and intestinal IL-4 in each intervention group were lower(P< 0. 05), and the co-culture group was lower than LC group and BN group(P< 0. 05). There was no significant difference between LC group and BN group(P> 0. 05). There were no significant differences in serum DAO, bacterial ectopic rate, sIgA, IL-2, IL-4 levels and IL-2/IL-4 levels between the co-culture group and the control group(P> 0. 05). The results showed that LC and BN co-culture supernatant can effectively regulate intestinal micro-ecology of AAD mice, improve intestinal mucosal barrier function, and improve intestinal and global immune function.