Prostaglandin (PG) E₂, a bioactive lipid biosynthesized from arachidonic acid, exerts its functions through 4 cognate receptors EP1-4. Our previous studies suggested that PGE₂ attenuates antitumor immunity in the tumor microenvironment by the recruitment and activation of regulatory T cells (Tregs), which are a subset of T cells specialized in immunosuppression. However, whether such actions of PGE₂ on Treg is a direct or indirect action remains largely unknown. To address this question, we generated induced Tregs (iTregs) from purified splenic CD4 ⁺ T cells by CD3/28 stimuli in the presence of TGF-β in vitro and examined the direct effect of PGE₂. Using flow cytometry analysis, we found that the expression level of Foxp3, a master transcription factor of Treg, and 4-1bb, a coactivator of Treg, were both significantly enhanced upon PGE₂ treatment. Furthermore, these effects were notably suppressed in the presence of EP4 antagonist, suggesting that PGE₂ contributes to Treg activity through the EP4 receptor. In addition, RNA-seq revealed that not only 4-1bb, but other Treg coactivator genes such as Ctla4, Gitr, and Ox40, were greatly increased in the PGE₂ treatment group. Given that previous studies reported that high expression level of these genes in intratumoral Tregs is positively correlated to Treg immune suppression activity, we speculated that PGE₂ produced in the tumor microenvironment may directly induce the activated phenotype of intratumoral Treg through EP4 receptor.