DNA adenine methylation (N⁶-methyl-2′-deoxyadenosine, m⁶dA) plays important functional roles in prokaryotes and protists, including regulation of gene transcription, DNA replication and repair, and the restriction-modification system. However, there is no definitive evidence supporting the presence of DNA adenine methylation in genomic DNA of higher eukaryotes, such as mammals and plants, where DNA cytosine methylation (5-methylcytosine) instead is well recognized as an important epigenetic mark that has regulatory roles in various biological processes. In the current study, we developed a Dpn I cleavage coupled with size-exclusion ultrafiltration method, with which we discovered the wide-spread existence of m⁶dA in genomic DNA of higher eukaryotes, including human cells, rat tissues, and plants besides bacteria and protists by employing high-resolution mass spectrometry analysis. And the contents of m⁶dA vary in different cell types with the range of 0.00006–0.00077% (m⁶dA dA⁻¹). Moreover, similar to N⁶-methyladenosine (m⁶A) in RNA, m⁶dA contents significantly decreased in type 2 diabetes mellitus (T2DM) patients compared to control subjects, indicating m⁶dA plays important roles in the pathogenesis of T2DM as m⁶A. In addition, knockdown of cellular fat mass and obesity-associated (FTO) protein increased the m⁶dA content, while overexpression of cellular FTO decreased m⁶dA content in DNA, suggesting m⁶dA and m⁶A may share the same demethylase of FTO. The demonstration of the universal presence of DNA adenine methylation constitutes the first and essential step toward understanding of m⁶dA functions in higher eukaryotes.