Objective To study the differences in intestinal flora of normal and type 2 diabetic mice, the effect of alcoholic extract of Coreopsis tinctoria Nutt on mouse intestinal flora, and explore the possible relationship between alcoholic extract of Coreopsis tinctoria Nutt, intestinal flora and type 2 diabetes mellitus (T2DM) in mice. Methods Stool samples were collected from the normal control group (A), high dose (1.8 g/kg) (B) and moderate dose (1.2 g/kg) (C) alcoholic extract of Coreopsis tinctoria Nutt model groups, metformin (0.2 g/kg) treatment group (D) and blank control (E) group. 16S rDNA real-time quantitative PCR assay was used to determine the levels of Clostridium coccoides and Bacteroides thetaiotaomicron in the stool samples. Pearson analysis method was used to analyze the correlation between the levels of target bacterial species and the fasting blood glucose (FBG) in the mice. Results 1. Compared with the normal control group, the levels of Clostridium coccoides and Bacteroides thetaiotaomicron in the T2DM model group were significantly lowered (P=0.017, P=0.002). 2. Compared with the model group, the levels of Clostridium coccoides and Bacteroides thetaiotaomicron of the high dose Coreopsis tinctoria Nutt alcohol extract group were significantly different (2 weeks:P=0.027, P=0.006; 4 weeks: P=0.007, P=0.012). 3. The levels of Clostridium coccoides and Bacteroides thetaiotaomicron were positively correlated with the FBG level in the mice. Conclusions The alcohol extract of Coreopsis tinctoria Nutt has certain effect on the intestinal flora in type 2 diabetic mice and there is certain correlation between the effect of alcohol extract of Coreopsis tinctoria Nutt and their blood glucose level.