The H3K9me2 histone methyltransferases G9a and GLP repress Mage-a class cancer germ-line (CG) antigen gene expression in murine embryonic stem (ES) cells, but the role of these enzymes in CG antigen gene regulation in human cancer cells is unknown. Here we show that whereas independent or dual knockdown of G9a and GLP in human cancer cells leads to reduced global and CG antigen promoter-associated H3K9me2 levels, it does not activate CG antigen gene expression. Moreover, CG antigen gene repression is maintained following pharmacologic targeting of G9a or treatment of G9a knockdown cells with the histone deacetylase inhibitor trichostatin A. However, G9a knockdown cells display increased sensitivity to CG antigen gene activation mediated by the DNA methyltransferase inhibitor decitabine. To account for these findings, we examined DNA methylation at CG antigen gene promoters in both cell types. We found robust DNA hypomethylation in G9a/GLP targeted murine ES cells but a lack of DNA methylation changes in G9a/GLP targeted human cancer cells; intriguingly, this distinction also extended to markers of global DNA methylation. These data reveal that G9a/GLP is required for DNA methylation of CG antigen genes and genomic DNA in murine ES cells, but not human cancer cells, and implicate DNA methylation status as the key epigenetic mechanism involved in CG antigen gene repression.