Abstract Background Valproic acid (VPA) is a first-line antiepileptic drug for glioblastoma (GBM) patients. There is also some evidence it improves the clinical outcome in those patients. However, the exact mechanism of VPA action is vague. Temozolomide (TMZ) is a first-line chemotherapeutic in GBM. Epigenetics offers a connection between genetic and environmental factors that influence the development of the disease. The best-characterized epigenetic mark is 5-methylcytosine (m5C) in DNA. The aim of that project is to show the effects of VPA administration on the total DNA methylation level. Material and Methods Using the nucleotide post-labeling method, we analyzed the total amount of m5C in DNA of GBM (T98G, U118, U138), cancer (HeLa) and normal (HaCaT) cell lines treated with VPA, and a combination of VPA and with TMZ. Results We adjusted the VPA doses to the ones achieved in the central nervous system during treatment. We observed dose-dependent changes in the total DNA methylation in neoplastic cell lines and the lack of such effect in a normal cell line. VPA alone produced a clear dose-dependent increase in total DNA methylation in GBM cell lines and scarce in the non-neoplastic cell line. In GBM cell lines, TMZ decreased the level of m5C. However, the exposition of GBM cells to the combination of VPA and TMZ caused an adverse synergistic effect resulting in DNA demethylation. The highest loss of m5C was observed at the highest concentration of TMZ (100 μM) and VPA (350 μM), after 3 h of incubation in the T98G cell line. Conclusion Total DNA methylation changes in glioma cell lines under VPA treatment suggest the new mechanism of that drug action and promote clinical implications for adjusting VPA and TMZ therapy in GBM patients. Our results show the potential and possible obstacles of the combined therapy of TMZ with VPA. Our experiments show that combined therapy with both drugs leads to total DNA hypomethylation. Therefore the conclusion would be to stop VPA administration during TMZ chemotherapy temporarily.