Inflammatory bowel diseases (IBDs) are characterized by unrestrained innate and adaptive immune responses and compromised intestinal epithelial barrier integrity. Regulatory T (T(reg)) cells are crucial for maintaining self-tolerance and immune homeostasis in intestinal tissues. Prostaglandin E(2) (PGE(2)), a bioactive lipid compound derived from arachidonic acid, can modulate T cell functions in a receptor subtype-specific manner. However, whether PGE(2) regulates T(reg) cell function and contributes to IBD pathogenesis remains unclear. Here, we found that the PGE(2) receptor subtype 2 (EP2) is highly expressed in T(reg) cells. T(reg) cell-specific deletion of EP2 resulted in increased T(reg) cell numbers, and enhanced granzyme B(GzmB) expression and immunosuppressive capacity of T(reg) cells in mice. Adoptive transfer of EP2-deficient T(reg) cells attenuated naive CD4(+) T cell transfer-induced colitis in Rag1(-/-) mice. Mice with EP2-deficient T(reg) cells were protected from 2,4,6-trinitrobenzene sulfonic acid (TNBS)- and dextran sodium sulfate (DSS)-induced colitis. Pharmacological blockage of EP2 with PF-04418948 markedly alleviated DSS-induced colitis in mice in a T(reg)-dependent manner. Mechanistically, activation of EP2 suppressed T(reg) cell function, at least in part, through reduction of GzmB expression via PKA-mediated inhibition of NF-kappaB signaling. Thus, we identified the PGE(2)/EP2 axis as a key negative modulator of T(reg) cell function, suggesting EP2 inhibition as a potential therapeutic strategy for IBD treatment.