PurposeWe have previously shown that TRDMT1 methyltransferase is a regulator of chemotherapy-associated responses in glioblastoma cells. Despite the fact that glioblastoma, a common and malignant brain tumor, is widely characterized in terms of genetic and epigenetic markers, there are no data on TRDMT1-related changes in 5-methylcytosine pools in the genome. In the present study, the effect ofTRDMT1gene knockout (KO) on DNA methylome was analyzed.MethodsCRISPR-based approach was used to obtainTRDMT1KO glioblastoma cells. Total 5-methylcytosine levels in DNA, DNMT1 pools and DNMT activity were studied using ELISA. Reduced representation bisulfite sequencing (RRBS) was considered to comprehensively evaluate DNA methylome in glioblastoma cells withTRDMT1KO.ResultsTRDMT1KO cells were characterized by decreased levels of total 5-methylcytosine in DNA and DNMT1, and DNMT activity. RRBS-based methylome analysis revealed statistically significant differences in methylation-relevant DMS-linked genes in control cells compared toTRDMT1KO cells.TRDMT1KO-associated changes in DNA methylome may affect the activity of several processes and pathways such as telomere maintenance, cell cycle and longevity regulating pathway, proteostasis, DNA and RNA biology.ConclusionsTRDMT1 may be suggested as a novel modulator of gene expression by changes in DNA methylome that may affect cancer cell fates during chemotherapy. We postulate that the levels and mutation status ofTRDMT1should be considered as a prognostic marker and carefully monitored during glioblastoma progression.Supplementary InformationThe online version contains supplementary material available at 10.1007/s11060-023-04304-8.