GLP-1 analogue exendin-4, a glucagon-like peptide 1 receptor (GLP-1R) agonist which shares 53% sequence with GLP-1, plays an essential role in human tumors. However, the function and mechanisms underlying the effects of exendin-4 on glioma cell migration, invasion and epithelial-to-mesenchymal transition are still obscure. Firstly, we demonstrated that GLP-1R was expressed in all glioma cell lines including U87, U251, U373 and A172. Exendin-4 treatment inhibited glioma cell survival, proliferation, migration and invasion. Also, exendin-4 inhibited epithelial-to-mesenchymal transition through positively regulating the expression of E-cadherin (epithelial marker), and negatively regulating the level of Vimentin (mesenchymal marker). Interestingly, we next demonstrated that exendin-4 elevated sirt3 expression dependent on the high level of GLP-1R in U87 and 251 cells. Finally, we confirmed that depletion the level of GLP-1R or sirt3 both reversed the inhibitory action of exendin-4 on glioma cell migration and invasion. These findings demonstrate that exendin-4 treatment suppressed the migration and invasion of glioma cells through GLP-1R/sirt3 pathway and exendin-4 plays an inhibitory effect on glioblastoma cell migration and invasion.