DNA methylation is an essential epigenetic modification. In bacteria, it is involved in gene regulation, DNA repair, and control of cell cycle. In eukaryotes, it acts in concert with other epigenetic modifications to regulate gene expression and chromatin structure. In addition to these biological roles, DNA methyltransferases have several interesting applications in biotechnology, which are the main focus of this review, namely, (1) in vivo footprinting: as several bacterial DNA methyltransferases cannot methylate DNA bound to histone proteins, the pattern of DNA methylation after expression of DNA methyltransferases in the cell allows determining nucleosome positioning; (2) mapping the binding specificity of DNA binding proteins: after fusion of a DNA methyltransferase to a DNA-binding protein and expression of the fusion protein in a cell, the DNA methylation pattern reflects the DNA-binding specificity of the DNA-binding protein; and (3) targeted gene silencing: after fusion of a DNA methyltransferase to a suitable DNA-binding domain, DNA methylation can be directed to promoter regions of target genes. Thereby, gene expression can be switched off specifically, efficiently, and stably, which has a number of potential medical applications.