Abstract: Our previous report demonstrated puerarin protected β cells by up-regulating the expression of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R). However, whether the anti-diabetic effects of puerarin in vivo depend on GLP-1R activation has not been clarified. In this study, the GLP-1R agonist exendin-4 (Ex4) and the GLP-1R antagonist exendin 9-39 (Ex9-30) were used. Type 2 diabetes was induced in C57BL/6J mice by a high fat diet (HFD) and divided into the following groups: control, HFD, HFD/puerarin (300 mg·kg^-1·d^-1), HFD/puerarin/exendin 9-39 (Ex9-39: 10 nmol·kg^-1·d^-1), and HFD/puerarin/exendin-4 group (Ex4: 10 nmol·kg^-1·d^-1). Animal experiments were approved by the Research Animal Care Committee of Affiliated Hospital of Integrated Traditional Chinese and Western Medicine, Nanjing University of Chinese Medicine (AEWC-025). Puerarin was administered orally, Ex9-39 and Ex4 were administered by intraperitoneal injection for 10 days. Compared with HFD group, after 10-day treatment, the fasting blood glucose and oral glucose tolerance test (OGTT) of diabetic mice were effectively improved by puerarin (P<0.05). Meanwhile, serum insulin levels were increased by puerarin, and levels of glucagon, triglycerides, and total cholesterol were significantly reduced (P<0.05). Importantly, Ex4 significantly enhanced the anti-diabetic effects of puerarin in HFD mice, while Ex9-39 markedly inhibited the effects of puerarin (P<0.05), which indicates that the effects of puerarin depend on GLP-1R activation. Furthermore, results of Western blotting of liver tissue showed puerarin effectively activated AKT and inhibited FOXO1, which relied on GLP-1R activation as well. Taken together, our findings demonstrate that puerarin ameliorates glucose homeostasis in HFD mice and is dependent on GLP-1R activation. This study provides experimental support for the potential application of puerarin.