This study investigates the effects Dioscorea opposita polysaccharides (DOP) on insulin resistance, lipid metabolism, oxidative stress, and intestine microbiota in high-fat-diet and streptozotocin induced type 2 diabetes (T2DM) rats. Low dose (DOP-L, 200 mg/kg BW), high dose (DOP-H, 400 mg/kg BW) and D. opposita powder (DO, 400 mg/kg BW) were oral-administrated to T2DM rats. After 6 weeks of treatment, supplementation of DOP-H and DO improved body weight and glucose/lipid metabolism-related indicators, including glucagon-like peptide 1, total cholesterol and high-density lipoprotein cholesterol. DOP-H and DO suppressed liver oxidative stress through increasing the level of superoxide dismutase, catalase, glutathione and reducing malondialdehyde. DOP attenuated the pathological change in liver, such as hepatic steatosis, and thus improved the liver function. Furthermore, the anti-diabetic effects of DOP was correlated with alterations of the gut microbiota, including an increase in Firmicutes and Bacteroidetes and a decrease in Actinobacteria and Proteobacteria, which promoted a healthier gut environment. Further analysis of short-chain fatty acids and metabolites provided evidences of DOP's regulatory effects on cecal contents in T2DM rats. Therefore, DOP-H present decent effects on T2DM, suggesting that DOP can ameliorate the insulin resistance and restore blood lipid level of T2DM rats with high-fat-diet by regulating intestinal microbiota.