Cyclooxygenase (COX)-2 and its product prostaglandin (PG) E₂ underlie an immunosuppressive network that is important in the pathogenesis of non–small cell lung cancer. CD4⁺CD25⁺ T regulatory (Treg) cells play an important role in maintenance of immunologic self-tolerance. CD4⁺CD25⁺ Treg cell activities increase in lung cancer and appear to play a role in suppressing antitumor immune responses. Definition of the pathways controlling Treg cell activities will enhance our understanding of limitation of the host antitumor immune responses. Tumor-derived COX-2/PGE₂ induced expression of the Treg cell-specific transcription factor, Foxp3, and increased Treg cell activity. Assessment of E-prostanoid (EP) receptor requirements revealed that PGE₂-mediated induction of Treg cell Foxp3 gene expression was significantly reduced in the absence of the EP4 receptor and ablated in the absence of the EP2 receptor expression. In vivo, COX-2 inhibition reduced Treg cell frequency and activity, attenuated Foxp3 expression in tumor-infiltrating lymphocytes, and decreased tumor burden. Transfer of Treg cells or administration of PGE₂ to mice receiving COX-2 inhibitors reversed these effects. We conclude that inhibition of COX-2/PGE₂ suppresses Treg cell activity and enhances antitumor responses.