Regulatory T cells (Treg cells), a subgroup of CD4+ lymphocytes, play a crucial role in serving as an immune suppressor and in maintaining peripheral tolerance. As the accumulation of Treg cells in the tumor microenvironment is significantly associated with a decreased survival time of patients, they are considered as an important therapeutic target in the immunotherapy of human cancers. These cells are either derived from the thymus, which are called (CD4CD25hiCD127low) natural Treg cells (nTreg cells), or they are generated from CD4+CD25− naive T cells by transforming growth factor-beta 1 and interleukin 2 (IL-2) in the periphery, which are called induced Treg cells (iTreg cells). Although iTreg cells are unstable, nTreg cells stably express forkhead box P3 (FOXP3) protein. Moreover, nTreg cells can be classified as memory (CD45RA−) and naive (CD45RA+) Treg cells, and this classification is based on the expression of CD45RA. FOXP3, which is a master regulator transcription factor, is essential for the functions of Treg cells, and it is mainly controlled by epigenetic mechanisms. The cyclooxygenase 2 (COX2)/prostaglandin E2 (PGE2) pathway is also reported to contribute to the regulatory functions of tumor-infiltrating Treg cells. As a new approach, we investigated whether S-adenosylmethionine (SAM), a substrate of DNA methyltransferase, attenuates the immune-suppressive capacity of the naive subtype of nTreg cells (CD4CD25hiCD127loCD45RA+). Moreover, we examined the effects of PGE2/COX2 pathway blockers on the suppressive capacity of Treg cells. We found that SAM diminished the suppression competency of Treg cells by decreasing the FOXP3 mRNA and protein levels in a dose-dependent manner. SAM increased the DNA methylation of FOXP3 at the first intron site. In addition, SAM decreased the mRNA and protein levels of the IL-10 cytokine, which has suppressive roles in the immune system. Moreover, mRNA levels of interferon gamma (IFNG) were found to be increased. COX2 inhibition and blockage of PGE2 receptors also reduced the protein and mRNA levels of IL-10, but they did not exhibit any significant effect on Treg cells’ suppression in the coculture system. Our results show that SAM might be considered and investigated as a promising agent for immunotherapy in the future.