Intestinal microbiota plays an important role in maintaining the microecological balance of the gastrointestinal tract in various animals. Disturbances in the intestinal microbiota may lead to the proliferation of potentially pathogenic bacteria that become the dominant species, leading to intestinal immune disorders, intestinal inflammation, and other intestinal diseases. Numerous studies have been confirmed that high-altitude exposure affects the normal function of the intestine and the composition of the intestinal microbiota. However, it is still necessary to reveal the changes in intestinal microbiota in high-altitude exposure environments, and clarify the relationship between the proliferation of potentially pathogenic bacteria and intestinal injury in this environment. In addition, explored probiotics that may have preventive effects against intestinal diseases.C57BL/6 mice were randomly divided into three groups, a high-altitude group (HA), control group (C), and high-altitude probiotic group (HAP). The HA and HAP groups were subjected to hypoxia modeling for 14 days in a low-pressure oxygen chamber with daily gavage of 0.2 mL of normal saline (HA) and Lactobacillus johnsonii YH1136 bacterial fluid (HAP), while the control group was fed normally. L. johnsonii YH1136 was isolated from feces of a healthy Tibetan girl in Baingoin county, the Nagqu region of the Tibet Autonomous Region, at an altitude of 5000 meters. Our observations revealed that gavage of YH1136 was effective in improving the damage to the intestinal barrier caused by high-altitude exposure to hypoxic environments and helped to reduce the likelihood of pathogenic bacteria infection through the intestinal barrier. It also positively regulates the intestinal microbiota to the extent of Lactobacillus being the dominant microbiome and reducing the number of pathogenic bacteria. By analyzing the expression profile of ileal microRNAs and correlation analysis with intestinal microbiota, we found that Staphylococcus and Corynebacterium1 cooperated with miR-196a-1-3p and miR-3060-3p, respectively, to play a regulatory role in the process of high-altitude hypoxia-induced intestinal injury.These findings revealed the beneficial effect of L. johnsonii YH1136 in preventing potential endogenous pathogenic bacteria-induced intestinal dysfunction in high-altitude environments. The mechanism may be related to the regulation of intestinal injury from the perspective of the gut microbiota as well as miRNAs.Copyright © 2022 Wan, Zhang, Jia, Qin, Sun, Xin, Zeng, Jing, Fang, Pan, Zeng, Bai, Wang, Ma and Ni.