Abstract: Metabolic syndrome characterized by obesity, hyperglycemia and liver steatosis is becoming prevalent all over the world. Herein, a water insoluble polysaccharide (WIP) was isolated and identified from the sclerotium of Poria cocos, a widely used Traditional Chinese Medicine. WIP was confirmed to be a (1-3)-β-D-glucan with an average Mw of 4.486×10⁶ Da by NMR and SEC-RI-MALLS analyses. Furthermore, oral treatment with WIP from P. cocos significantly improved glucose and lipid metabolism and alleviated hepatic steatosis in ob/ob mice. 16S DNA sequencing analysis of cecum content from WIP-treated mice indicated the increase of butyrate-producing bacteria Lachnospiracea, Clostridium. It was also observed that WIP treatment elevated the level of butyrate in gut, improved the gut mucosal integrity and activated the intestinal PPAR-γ pathway. Fecal transplantation experiments definitely confirmed the causative role of gut microbiota in mediating the benefits of WIP. It is the first report that the water insoluble polysaccharide from the sclerotium of P. cocos modulates gut microbiota to improve hyperglycemia and hyperlipidemia. Thereby, WIP from P. cocos, as a prebiotic, has the potential for the prevention or cure of metabolic diseases and may elucidate new mechanism for the efficacies of this traditional herbal medicine on the regulation of lipid and glucose metabolism.