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Ophiopogonis Radix (Maidong in Chinese), the tuberous roots of Ophiopogon japonicus having polysaccharide as the main active ingredient, is a traditional Chinese herb and popularly used as functional food additive in China. Here, a homogeneous polysaccharide fraction (OJP-W1) was isolated and characterized from Ophiopogonis Radix collected from Sichuan (Chuan-Maidong). The therapeutic effects of OJP-W1 in diabetes symptoms, gut microbiota and metabolism of short-chain fatty acids (SCFAs) in high-fat diet (HFD)-induced type-2 diabetes mice were investigated. Results showed that OJP-W1 significantly ameliorated HFD-induced insulin resistance and glucose tolerance. Meanwhile, OJP-W1 regulated the gut microbiota dysbiosis in HFD-fed mice, as indicated by increasing Actinobacteria and Bifidobacterium, decreasing Proteobacteria and type-2 diabetes-enriched taxa (e.g. Desulfovibrionaceae, Dorea and Ruminococcaceae). In addition, OJP-W1 could improve the metabolic disorder of SCFAs in HFD-fed mice. This study indicated that OJP-W1 might serve as a novel functional food in preventing diabetes-associated gut microbiota dysbiosis and SCFA metabolic disorder.