Visitors to high altitude are susceptible to hypoxia-induced acute intestinal mucosal barrier injury and severe gastrointestinal disorders, which are life-threatening. Citrus tangerine pith extract (CTPE) is rich in pectin and flavonoids and has been proved to enhance intestinal health and improve gut dysbiosis. In this study, we aim to explore the protective effect of CTPE on ileum injury induced by intermittent hypobaric hypoxia in a mouse model. Balb/c mice were divided into blank normoxia (BN), blank hypobaric hypoxia (BH), hypobaric hypoxia plus CTPE (TH), and hypobaric hypoxia plus Rhodiola extract (RH) groups. From the 6th day of gavage, mice in BH, TH, and RH groups were transferred into a hypobaric chamber at a simulated elevation of 6000 m for 8 hours per day for 10 days. Then half the mice were tested for small intestine movement, and others were used to evaluate intestinal physical barrier function, inflammation, and gut microbiota. Results showed that CTPE reversed the increase of intestinal peristalsis, effectively attenuated impaired structural integrity of ileum, improved the mRNA and protein expression levels of tight junction proteins, and reduced serum D-LA content in mice to alleviate hypoxia-induced mucosal barrier damage. Moreover, CTPE supplementation ameliorated hypoxia-induced intestinal inflammation response by significantly downregulating the proinflammatory cytokines IL-6, TNF-α and IFN-γ. By 16S rDNA gene sequencing of gut microbiota, CTPE significantly increased the abundance of probiotic Lactobacillus, suggesting that CTPE may be used as a prebiotic to regulate ecology of intestinal microorganisms. In addition, Spearman rank correlation analysis revealed that changed gut microbiota were significantly correlated with alteration of intestinal barrier function indexes. Taken together, these results indicate that CTPE effectively alleviates hypoxia-induced intestinal injury in mice and enhances intestinal integrity and barrier function by altering intestinal microbiota composition.