Longan pulp polysaccharide is a bioactive component with prebiotic activity and intestinal barrier protection. This study aimed to evaluate the influence of digestion and fermentation on the bioavailability and intestinal barrier protection of polysaccharide LPIIa from longan pulp. The molecular weight of LPIIa didn’t change significantly after gastrointestinal digestion in vitro. After fecal fermentation, 56.02% of LPIIa was consumed by gut microbiota. The short-chain fatty acid level in LPIIa group was 51.63% higher than that in blank group. LPIIa intake also increased short-chain fatty acid production and G-protein-coupled receptor 41 expression in the colon of mice. Moreover, LPIIa improved the relative richness of Lactobacillus, Pediococcus, and Bifidobacterium in colon content. Compared to LPIIa, fecal fermented LPIIa better protected intestinal epithelial barrier by increasing Zonula occludens-1 expression. These results provided an important basis for the design of functional food based on longan polysaccharides to prevent intestinal barrier damage related diseases.