ABCG2 is a member of the ATP-binding cassette transporter family, which has been detected in a wide variety of human aggressive tumors, including glioma stem cells (GSCs), glioma tissues of higher grades, and implanted glioma xenografts. Previous research has implied that ABCG2 might be associated closely with invasion and spread in tumors. However, the specific roles and mechanisms of ABCG2 in regulating the migration and invasion of GSCs remain unclear. In this study, we aimed to identify the effects and mechanisms of ABCG2 on invasion by GSCs. Our results showed that downregulation of ABCG2 protein significantly inhibited the migration and invasion potentials of U251 GSCs. Further research on the underlying mechanism showed that the effects of ABCG2 downregulation on inhibiting the migration and invasion of U251 GSCs were through significantly decreasing the activity of matrix metalloproteinase-9, but not the expression of matrix metalloproteinase-9 protein. These findings show that ABCG2 plays an important role in regulating the migration and invasion of GSC, and represents a potential novel therapeutic agent to target the progression of GSCs.