Epigenetic changes play a role in all major events during tumorigenesis and changes in glycan structures are hallmarks of virtually every cancer. Also, proper N-glycosylation of membrane receptors is important in cell to cell and cell–environment communication. To study how modulation of epigenetic information can affect N-glycan expression we analyzed effects of epigenetic inhibitors on HeLa cell membrane N-glycome. HeLa cells were treated with DNA methylation (zebularin and 5-aza-2-deoxycytidine) and histone deacetylation (trichostatin A and Na-butyrate) inhibitors. The effects on HeLa cell membrane N-glycome were analyzed by hydrophilic interaction high performance liquid chromatography (HILIC). Each of the four epigenetic inhibitors induced changes in the expression of HeLa cell membrane N-glycans that were seen either as an increase or a decrease of individual glycans in the total N-glycome. Compared to DNA methylation inhibitors, histone deacetylation inhibitors showed more moderate changes, probably due to their higher gene target selectivity. The results clearly show that composition of HeLa cell membrane N-glycome can be specifically altered by epigenetic inhibitors. Glycans on the cell membrane are essential elements of tumor cell's metastatic potential and are also an entry point for nearly all pathogenic microorganisms. Since epigenetic inhibitors used in this work are registered drugs, our results provide a new line of research in the application of these drugs as anticancer and antimicrobial agents. This article is part of a Special Issue entitled Glycoproteomics. ► Aberrant epigenetic profiles and aberrant N-glycosylation are involved in tumorigenesis. ► HeLa cells were treated with DNA methylation and histone deacetylation inhibitors. ► Hydrophilic interaction high performance liquid chromatography revealed membrane N-glycome. ► Epigenetic inhibitors either up- or down-regulated individual N-glycan structures. ► Applied epigenetic inhibitors are efficient modulators of the cell membrane N-glycome.