As major suppressors of inflammation, regulatory T cells (Tregs) maintain immune homeostasis and tolerance by means of various inhibitory molecules they express. In tumor, however, Tregs limit anti-tumor responses, thereby aggravating tumor progression. Understanding the regulatory mechanisms of Treg cell functions in tumor offers opportunities that enhance therapeutic effects of anti-tumor immunotherapy. IL-12 family cytokine IL-27 is a potent regulator of immune system, but the exact roles in anti-tumor immunity and in regulating Treg cells remain unclear. Using knock-out mice of IL-12 cytokine subunits and IL-27 receptor, we demonstrate that IL-27 has a crucial role in regulating the suppressive function of Treg cells, particularly in tumor microenvironment. Lack of IL-27 or IL-27 receptor induces delayed tumor growth with defective Treg functions in vivo. These findings unveil unprecedented role for IL-27 in regulating Treg cells, which may provide a novel target for breaking immunosuppressive milieu in tumor environment.