Polarization of naïve CD4 T cells into T helper type 2 (TH2) cells is characterized by expression of IL-4 and silencing of IFN-gamma. Here we show that during TH2 polarization, the DNA methyltransferase Dnmt3a is recruited to the IFN-gamma promoter and correspondingly the promoter undergoes progressive de novo methylation. Notably, the CpG located at the -53 position becomes methylated rapidly and this methylation inhibits ATF2/c-Jun and CREB transcription factor binding in vitro. In vivo, the same factors bind to the unmethylated IFN-gamma promoter in T helper type 1 (TH1) cells but not the methylated IFN-gamma promoter in TH2 cells. Furthermore, methylation at the -53 CpG alone is sufficient to inhibit the IFN-gamma promoter-driven reporter gene expression in a TH1 cell line. These findings suggest that rapid methylation of the evolutionarily conserved -53 CpG by Dnmt3a may suppress IFN-gamma transcription in developing TH2 cells by directly inhibiting transcription factor binding.