Among all brain tumors diagnosed in children, medulloblastomas (MBs) are associated with a poor prognosis. The etiology of MB is not fully understood, yet the impact of epigenetic alterations of oncogenes has previously been established. During the past decade, the human cytomegalovirus (HCMV) has been detected in several types of cancer, including MB. Since DNA methylation occurs in the cell nucleus and this is considered a host defence response, we studied the impact of HCMV infection on DNA methyltransferase (DNMT‑1) in MB (D324) cells, human umbilical vein endothelial cells (HUVECs) as well as in MB tissue sections. We hypothesized that infection and DNMT‑1 intracellular localization are linked. Uninfected and HCMV‑infected D324 cells and HUVECs were analyzed for HCMV immediate early (HCMV‑IE) protein, HCMV‑glycoprotein B (HCMV‑gB) and DNMT‑1 using immunofluorescence staining and quantitative ELISA. DNMT‑1 localized to the nucleus of uninfected and HCMV‑IE- expressing D324 cells and HUVECs, but accumulated in the extra nuclear space in all HCMV‑gB-positive cells. Inhibition of HCMV late protein expression by Cymevene® (ganciclovir) prevented the cytoplasmic localization of DNMT‑1. Treatment of HCMV‑ infected D324 cells and HUVECs with the methylation inhibitor 5-Azacytidine (5AZA), significantly increased HCMV‑IE and HCMV‑gB gene transcription and protein expression. Immunohistochemical staining of DNMT‑1 and HCMV proteins in MB cancer tissue sections revealed both nuclear and cytoplasmic DNMT‑1 localization. In conclusion, DNMT‑1 resides in the cytoplasm of HCMV‑gB-expressing HUVECs and D324 cells. Increased viral protein synthesis in 5AZA-treated cells suggests that HCMV replication may benefit from a DNA methyltransferase-free cellular environment. Our findings emphasize the importance of assessing potential viral activation in the treatment of MB patients with epigenetic drugs.