The forkhead family protein Foxp3 is a unique marker of regulatory T cells and plays a crucial role in the development and function of those cells. Ectopic expression of Foxp3 abolishes the expression of many cytokines in uncommitted cells but there is little information about whether it causes gene silencing in differentiated cells. In this study, we showed that ectopic expression of Foxp3 in primary T helper 2 cells abolished IL-4 gene expression. Foxp3 inhibited nuclear translocation of NFkappaB by increasing the stability of the NFkappaB inhibitor IkappaBalpha, which in turn reduced in vivo binding of NFkappaB to the IL-4 promoter region. Moreover, Foxp3 over-expression induced inactive chromatin structure by decreasing in vivo binding levels of acetylated histone 3 while increasing methylated histone 3 at lysine 9 in the IL-4 genomic locus. Our results suggest that Foxp3 could induce gene silencing by inhibiting NFkappaB activity and by causing its target loci to adopt an inactive chromatin configuration.