The DNA N6-methyladenine (6mA) modification is a prevalent epigenetic mark in prokaryotes, but the low abundance of 6mA in eukaryotes has recently received attention. The possible role of 6mA as an epigenetic mark in eukaryotes is starting to be recognized. This review article addresses the epigenetic roles of 6mA in eukaryotes. The existence of 6mA in metazoans and plants, the correlation of 6mA with gene expression, the enzymes catalyzing the deposition and removal of the 6mA modification, the relationship of 6mA to nucleosome positioning, the 6mA interaction with chromatin, its role in tumorigenesis and other physiological conditions/diseases and technical issues in 6mA detection/profiling and bioinformatics analysis are described. New directions and unresolved issues (e.g., the base-pair-resolution 6mA-sequencing method and gene activation vs. repression) in 6mA research are discussed. Copyright © 2020 Elsevier B.V. All rights reserved.