Regulatory T cells (Tregs) play a pivotal role in the maintenance of homeostasis between immune response and immune tolerance. The transcription factor Foxp3 and the surface protein CD25 are the two key molecules characterizing Tregs. In autoimmune and various other chronic inflammatory diseases, the expression of Foxp3 is severely down-regulated. However, the molecular mechanism underlying the down-regulation of Foxp3 is not understood yet. Because the IL-12p40 homodimer (p40(2)) is markedly up-regulated in response to various inflammatory stimuli, the present study was undertaken to explore the role of p40(2) in the regulation of Foxp3 in naive mouse splenocytes. IL-12p40(2) dose-dependently inhibited the expression of Foxp3 and CD25, but not CD4. Interestingly, this inhibition was absent in splenocytes of IL-12Rbeta1(-/-), but not IL-12Rbeta2(-/-), mice. Moreover, suppression of Foxp3 in wild-type and IL-12Rbeta2(-/-) splenocytes was accompanied by production of NO. Consistently, l-N(6)-(1-iminoethyl)-lysine hydrochloride, an inhibitor of inducible NO synthase (iNOS), and PTIO, a scavenger of NO, restored the expression of Foxp3 and CD25 in p40(2)-stimulated splenocytes, and p40(2) was unable to down-regulate Foxp3 and CD25 in splenocytes from iNOS(-/-) mice. Furthermore, NO, but not p40(2), was able to inhibit Foxp3 in purified CD4(+)CD25(+) T cells in the absence of iNOS-expressing cells. Hence, our results clearly demonstrate that p40(2) induces NO production via IL-12Rbeta1 and that NO subsequently suppresses Tregs in naive mouse splenocytes. This study, therefore, delineates an unprecedented biological function of p40(2) in the regulation of Foxp3 via IL-12Rbeta1-mediated NO production.