Regulatory T cells (Tregs) have been shown to play a crucial role in maintaining self-tolerance and suppressing autoimmunity. The forkhead transcription factor, FoxP3, is a key molecule necessary and sufficient for Tregs development and function. However, the molecular mechanisms by which FoxP3 regulates the phenotypic (anergic) and the functional (suppressive) characteristics of Tregs are not well defined. Here we found that the promoter DNA-binding activity of AP-1 transcription factors is selectively inhibited in the naturally occurring CD4+ CD25+ Tregs from mice. The impaired AP-1 DNA binding is not the result of the decreased nuclear translocation of AP-1 family transcription factors, including c-Jun, JunB, and c-Fos. FoxP3 significantly suppresses both the transcriptional activity and promoter DNA-binding of AP-1 by interacting with c-Jun. The N-terminus of FoxP3, but not its C-terminus forkhead domain, specifically interacts with phosphorylated c-Jun and alters c-Jun subnuclear distribution. This N-terminus of FoxP3 with nuclear localization signals (FoxP3N/NLS) is able to suppress AP-1 transcriptional activity. Ectopic expression of FoxP3N/NLS sufficiently induces the unresponsiveness of mouse primary CD4+ CD25- T cells, whereas the full-length FoxP3 is required for the suppressive functions of Tregs. These findings uncover one of the mechanisms underlying how FoxP3 maintains the unresponsiveness of Tregs.