The transcription factor Foxp3 plays a key role in CD4(+)CD25(+) regulatory T (Treg) cell function. A correlation has been shown between survival and the frequency of tumor-infiltrating Foxp3-positive Treg cells in cancer patients. However, few studies have characterized the regulation of Foxp3 expression and function in Treg cells, which are known to comprise distinct subsets, with different roles in the complex tumor microenvironment. Here, we show that significantly more Foxp3-positive Treg cells accumulated in gastric tumors. In addition, we found increased expression of Foxp3 protein per cell in tumor-infiltrating Treg cells. Moreover, elevated Foxp3 expression in tumor-infiltrating Treg cells was associated with the TNM stage in gastric cancer patients. Importantly, further investigation within the tumor microenvironment showed that expression of Foxp3 in Treg cells correlated with expression of cyclooxygenase-2 (COX-2) and prostaglandin E(2) (PGE(2)). Furthermore, Treg cells with higher levels of Foxp3 were able to suppress the proliferation of autologous CD4(+)CD25(-) T cells. The suppression of the effector T-cell response was reversed by COX inhibitors and PGE(2) receptor-specific antagonists. Our data demonstrate a mechanism by which tumor-infiltrating Treg cells with increased Foxp3 expression can mediate immune suppression via COX-2/PGE(2) production in the gastric cancer microenvironment. Thus, we provide new insights into overcoming regulatory T-cell activity, which may be beneficial for the treatment of human gastric cancer.