Ulcerative colitis (UC) is an incurable chronic inflammation of the enteric tract. The aim of this study was to investigate the protective effects of arabinogalactan from Lycium barbarum on DSS-induced chronic colitis. A homogeneous arabinogalactan was isolated and purified from L. barbarum, named LBP-3, which mainly consisted of arabinose and galactose with a molar ratio of 1.00 : 0.82. LBP-3 treatment remarkably alleviated body weight loss, histopathological damage and the overproduction of pro-inflammatory cytokines and enzymes in UC mice. Additionally, the intestinal barrier integrity was partially recovered by the up-regulated expression of MUC2 and tight junction proteins. Moreover, the gut microbiota shift was reversed by LBP-3 administration by enriching potential probiotic bacteria (e.g., Ruminococcaceae) and inhibiting the proliferation of harmful bacteria (e.g., Enterobacteriaceae). Furthermore, SCFAs, as major metabolites of LBP-3 fermentation by gut microbiota, were also promoted so as to maintain relatively favorable intestinal homeostasis. Overall, our findings suggested LBP-3 from L. barbarum could be a potential therapeutic candidate against UC via improving intestinal barrier function and partially restoring gut microbiota and its metabolites.