Fibulin-3 has been considered as a regulator of glioma cell invasion, but little is known about the molecules regulating fibulin-3 expression. Metformin, an oral antidiabetic drug in the biguanide class, is known to inhibit proliferation and metastasis in a variety of cancer cells. In the present study, we determined the effect of metformin on the expression of fibulin-3 in U251 Human glioma cells. Metformin potently suppressed U251 cell adhesion and invasion. Metformin inhibited the expression of fibulin-3 at the transcriptional level. Moreover, metformin abolished the protein expression of fibulin-3 in a concentration-dependent manner. Furthermore, this compound suppressed the expression of matrix metalloproteinase-2, a key effector of glioma cell invasion, regulated by fibulin-3. Taken together, our results suggest that metformin abolishes fibulin-3 expression and subsequently inhibits invasion of glioma cells.