Glioblastoma multiforme (GBM) is the most malignant glioma. In the current study, 149 astrocytoma gene expression datasets were classified by prediction analysis of microarray. Strikingly, disks large homolog 3 (DLG3), a membrane-associated guanylate kinase-family gene, had the highest score in the GBM subset. DLG3 mRNA expression is significantly down-regulated in GBM relative to normal tissue and grade II or grade III astrocytoma according to the results of real-time polymerase chain reaction, and its protein expression shows an obvious difference by immunohistochemistry. Further assays show that DLG3 over-expression induces mitotic cell cycle arrest and apoptosis, and it inhibits proliferation and migration. However, DLG3 over-expression has almost no affect on invasion. The DLG3 protein expression in human brain GBM tissue and its effects on GBM cell invasion were not expected. Our data suggest that DLG3 is down-regulated in this cancer type. To our knowledge, this is the first report to clearly demonstrate the possible involvement of DLG3 in GBM.