Abstract: This study is aimed to investigate the effects of prebiotics on the expression of occludin and cytokines in the intestinal mucosal barrier of stressed rats in a high altitude environment. Forty-eight rats were acutely stressed in a high altitude hypoxia environment (3848 m). Rats in the hypoxia control group (n = 24) and prebiotics group (n = 24) were stressed for 2, 4 and 6 days before harvesting the intestinal mucosa. The expression of the occludin protein in the intestinal mucosa was analyzed by immunohistochemistry and tumor necrosis factor-α (TNF-α) and interleukin-10 (IL-10) expression were measured by the enzyme-linked immune sorbent assay in ileum tissue. The high altitude hypoxia environment significantly reduced occludin protein and mRNA expression in the control group, but prebiotics treatment elevated its expression on the 4th day and 6th day. Prebiotics treatment also significantly alleviated changes in TNF-α and IL-l0 induced by high altitude hypoxic stress. Prebiotics may help to maintain the integrity of epithelial cells by regulating expression of the occludin protein and cytokines.