Evidence has shown that Notch signaling modulates CD4+CD25+ regulatory T-cells (Tregs). As transcription factor Foxp3 acts as a master molecule governing the development and function of Tregs, we investigated whether Notch signaling might directly regulate Foxp3 expression. Here, we provide evidence that Notch signaling can modulate the FOXP3 promoter through RBP-J- and Hes1-dependent mechanisms. A conserved RBP-J-binding site and N-box sites were identified within the FOXP3 promoter. We show that the Notch intracellular domain (NIC), the active form of Notch receptors, activates a reporter driven by the FOXP3 promoter. Dissection of the FOXP3 promoter revealed bipartite effects of the RBP-J-binding site and the N-boxes: the RBP-J-binding site positively, while the N-boxes negatively regulated the FOXP3 promoter activity. Moreover, in freshly isolated Tregs, NIC-RBP-J complex is bound to the FOXP3 promoter in Tregs. Our results suggest that Notch signaling might be involved in the development and function of Tregs through regulating Foxp3 expression.