Malignant glioma is the most common central nervous system tumor of adults and is associated with a significant degree of morbidity and mortality. Gliomas are highly invasive and respond poorly to conventional treatments. Gliomas, like other tumor types, arise from a complex and poorly understood sequence of genetic and epigenetic alterations. Epigenetic alterations leading to gene silencing, in the form of aberrant CpG island promoter hypermethylation and histone deacetylation, have not been thoroughly investigated in brain tumors, and elucidating such changes is likely to enhance our understanding of their etiology and provide new treatment options. We used a combined approach of pharmacologic inhibition of DNA methylation and histone deacetylation, coupled with expression microarrays, to identify novel targets of epigenetic silencing in glioma cell lines. From this analysis, we identified >160 genes up-regulated by 5-aza-2'-deoxycytidine and trichostatin A treatment. Further characterization of 10 of these genes, including the putative metastasis suppressor CST6, the apoptosis-inducer BIK, and TSPYL5, whose function is unknown, revealed that they are frequent targets of epigenetic silencing in glioma cell lines and primary tumors and suppress glioma cell growth in culture. Furthermore, we show that other members of the TSPYL gene family are epigenetically silenced in gliomas and dissect the contribution of individual DNA methyltransferases to the aberrant promoter hypermethylation events. These studies, therefore, lay the foundation for a comprehensive understanding of the full extent of epigenetic changes in gliomas and how they may be exploited for therapeutic purposes.