The deposition of DNA methylation at promoters of transposons, X-linked genes, imprinted genes, and other lineage-specific genes is clearly associated with long-term transcriptional silencing. Thus, DNA methylation represents a key layer of epigenetic information in mammals that is required for embryonic development, germline differentiation, and, as shown more recently, for the function and maturation of neuronal tissues. The DNMT3A, DNMT3B, and DNMT3L proteins are primarily responsible for the establishment of genomic DNA methylation patterns and, as such, play an important role in human developmental, reproductive, and mental health. Progress in our understanding of this important protein family has been rapid in recent years and has been accompanied by stunning developments in the analysis of the human DNA methylome in multiple cell types. This review focuses on recent developments in the characterization of the DNMT3 family of DNA methyltransferases at the biochemical, structural, and functional levels. Interconnections between the DNA-based and histone-based layers of epigenetic information are particularly highlighted, as it is now clear that de novo methylation occurs chiefly in the context of nucleosomal templates.