Glioblastoma multiforme (GBM) has nearly uniformly fatal with a median survival of less than 2 years. While there have not been any novel anti-GBM therapeutics approved for many years, there has been the gradual accumulation of clinical data suggesting that the widely used anti-convulsant agent, valproic acid (VPA) may significantly prolong survival in GBM patients. This pre-clinical study aimed to determine the potential clinical utility of VPA in the treatment of GBM. Primary GBM cells were treated with VPA as a monotherapy and in combination with temozolomide and irradiation. At clinically achievable concentrations, VPA was shown to be effective as a monotherapy agent in the five primary lines tested. VPA was then used as a sensitizing agent to in vitro radiation and showed significant augmentation of in vitro irradiation therapy. In addition, when VPA, radiation and temozolomide were combined an additive, rather than synergistic effect was noted. Gene expression profiling demonstrated close clustering of triple treated cells with VPA mono-treated cells while untreated cells clustered closer with TMZ-irradiation dual treated cells. These microarray data suggest a dominant role of VPA at the gene expression level when combining these different treatment options. Moreover, in an in vivo tumor transplantation model, we were able to demonstrate an increase in animal survival when cells were pre-treated with irradiation-VPA and when triple treated. These findings provide a significant rationale for the investigation of VPA in the treatment of GBM patients.