Setdb1/Eset, a histone H3-lysine 9 (H3K9)-specific methyltransferase, interacts with various transcription factors to modify chromatin at target genomic regions. In this study, we provide evidence supporting that Setdb1 also regulates genomic DNA methylation. Whereas 5-methylcytosine (5-mC) signal majorly localizes at pericentric heterochromatin regions in wild type cells, knocking down Setdb1 resulted in diffuse localization of 5-mC signal. Setdb1 knockdown also reduced the expression and subcellular localization of the DNA methyltransferases Dnmt1 and Dnmt3a. Bisulfite mutagenesis studies revealed that Setdb1 knockdown reduced the methylation of satellite DNA sequences (heterochromatin) but not of intracistronic A particle (IAP) sequences (euchromatin). Our results suggest that Setdb1 directly or indirectly regulates the expression of DNA methyltransferases and thus affects DNA methylation and the structural organization of heterochromatin.