Malignant gliomas (MGs) are cancers with poor prognosis and limited therapeutic options. Herpes Simplex virus-1 thymidine kinase expressed from adenoviruses with prodrug ganciclovir (TK/GCV) is the best-characterized suicide gene therapy, whereas temozolomide (TMZ) is the first-line chemotherapy for MG. However, the potential of their combination has not been studied thoroughly. The aim of this study was to evaluate the therapeutic response of this combination and to study whether addition of valproic acid (VPA) could benefit the treatment outcome. Efficacies of different treatments were first studied in vitro in BT4C rat MG cells. Therapeutic assessment in vivo was done in an immunocompetent rat MG model for treatment efficacy and toxicity. In vitro, VPA was able to significantly enhance cytotoxicity and increase adenovirus-mediated transduction efficiency up to sevenfold. In vivo, rats receiving TK/GCV+TMZ had notably smaller tumors and enhanced survival (P<0.001) in comparison with control rats. However, VPA was not able to further enhance the treatment response in vivo. Leukocytopenia and thrombocytopenia were the major side effects. We conclude that careful optimization of the treatment schedules and doses of individual therapies are necessary to achieve an optimal therapeutic effect with TK/GCV+TMZ combination. No further in vivo benefit with VPA was observed.