Malignant gliomas are the most common type of primary brain tumors affecting 16,000 new patients every year in the United States. In this study, we undertook a systematic large-scale transcriptomics data mining study of 9,783 Affymetrix samples from the Genesapiens database (www.genesapiens.org) in order to identify the most glioma-specific biomarkers. We searched for genes that were highly expressed in 322 Glioblastoma Multiforme (GBM) samples and in 66 anaplastic astrocytomas as compared to 425 samples of the normal central nervous system as well as all other normal and cancerous tissues in the database. Transcription cofactor HES6 (Hairy and enhancer of split 6) emerged as one of the most glioma-specific genes. Since the role of HES6 in glioma pathogenesis is poorly understood, we chose to validate its expression by immunostaining and functional role by siRNA knockdown studies in glioma cell lines. HES6 protein levels were studied in a glioma tissue microarray material that consisted of 414 samples as well as in normal brain tissue controls. Positive HES6 immunoreactivity was present in 99 % of available gliomas. Recurrent tumors of grade 2 astrocytomas and grade 2-3 oligodenrogliomas showed higher levels of HES6 immunoreactivity than the corresponding primary tumors. Endothelial cells within the tumors were also stained in 75 % of gliomas. In functional studies, cell viability was reduced by 60 % and Caspase 3/7 activity elevated after HES6 silencing by RNA interference in A172 and LN405 cells. HES6 silencing also increased apoptosis 2 fold in two cell lines as measured by Apo-ONE Homogeneous Caspase-3/7 Assay. The biological role and consequences of HES6 silencing was explored with genome-wide analyses following RNAi, which indicated a key role for HES6 in e.g. p53, c-myc, and CREB1 transcriptional networks. Gene ontology analysis implicated genes involved in cellular movement, development and RNA post-transcriptional modification. HES6 protein was localized to the PML-bodies by immunostaining and co-localized with the creb binding protein (CBP). In conclusion, these results pinpointed HES6 as a potential therapeutic target playing a critical role in sustaining glioma cell growth, survival and possibly invasion. HES6 may also be a useful biomarker for gliomas. This work was supported by funding under the 6th FP of the European Union, Project RIGHT (LSHB-CT-2004-005276), and the Academy of Finland.