Forkhead box protein 3 (Foxp3) is indispensable for the development of CD4⁺CD25⁺ regulatory T cells (Tregs). Here we analyzed three prominent evolutionary conserved regions (ECRs) upstream of the transcription start site of the human FOXP3 gene. We show that ECR2 and ECR3 fragments derived from positions −1.3 to −2.0kb and −5.0 to −6.0kb, respectively, display basal transcriptional activity. Reporter constructs derived from ECR1, located between −0.6 and +0.23kb and thus the most proximal ECR in respect of transcription initiation, remained almost inactive. However, ECR1 was transactivated by the NF-κB subunit p65 in HEK 293 cells. In Jurkat and primary T cells, in addition to p65, a second stimulus delivered by either T-cell receptor stimulation or addition of PMA was needed. Co-expression of IκBα inhibited p65-mediated FOXP3 proximal promoter transactivation, and the NF-κB inhibitor curcumin reduced Foxp3 neoexpression in IL-2/CD3/CD28/TGF-β stimulated PBMCs. Moreover, proximal FOXP3 promoter transactivation was inhibited by Foxp3 and the SP transcription factor family member SP3. Thus, the human proximal FOXP3 promoter is controlled by activation through the TCR involving PKC and the NF-κB subunit p65 and by inhibition through a negative feedback loop and SP3.