Abstract: Objectives: To investigate the regulatory effect of ginger polysaccharides on the intestinal flora of mice with type 2 diabetes (T2D) induced by the combination of a high-fat diet (HFD) and low-dose streptozotocin (STZ). Methods: Polysaccharides were extracted from ginger by hot water extraction and ethanol precipitation. Single-factor experiments and an orthogonal experiment were used to optimize the extraction conditions, and then the best deproteinization method was selected to obtain ginger polysaccharide (GP). The scavenging ability of GP to 2,2'-diazo-bis-3-ethylbenzothiazoline-6-sulfonic diammonium salt (ABTS⁺) free radicals and hydroxyl radicals evaluated its antioxidant capacity in vitro. Subsequently, a type 2 diabetes mouse model was established by a high-fat diet and STZ. The fasting blood glucose (FBG) concentration and glycated serum protein (GSP) level of mice in each group were measured. The composition of the intestinal flora of diabetic mice was analyzed by 16S rRNA high-throughput sequencing technique. Results: The optimum extraction process of GP was as follows: The ratio of material to water was 1:20 g/mL, the extraction temperature was 90 ℃, the extraction time was 1 h, and Sevag reagent was used 5 times to deproteinize to obtain GP, the yield was 2.91%±0.25%, of which the total sugar content was 43.44%±0.99%. At the concentration of 4 mg/mL in GP, the scavenging rates of ABTS⁺ free radical and hydroxyl free radical by GP were 66.09% and 65.73% respectively. After the intervention of GP, FBG concentration and GSP level in the diabetic mice were significantly reduced (P<0.05). The 16S rRNA sequencing results showed that GP altered the gut microbiota composition and relative abundance in diabetic mice, and the relative abundance of beneficial bacteria such as Adlercreutzia, Akkermansia and Lactobacillus increased, while the relative abundance of harmful bacteria such as Prevotella decreased. Conclusions: Ginger polysaccharides obtained by hot water extraction and Sevag deproteinization could alter the composition of the intestinal flora in diabetic mice. The results of this study would provide a theoretical basis for ginger polysaccharide in the prevention and treatment of T2D.