Regulatory T (Treg) cells induce immune tolerance by suppressing host immune responses against self- or non-self-antigens, thus playing critical roles in preventing autoimmune diseases. However, tumor cells may take advantage of Treg cells to protect themselves from immune attack elicited by vaccines. Recent studies demonstrate the presence of Treg cells in various types of cancers and their suppressive function. Therefore, Treg cells at tumor sites have detrimental effects on immunotherapy directed to cancer and infectious diseases. This review will discuss antigen specificity of Treg cells, the factors that contribute to Treg cell generation and suppressive function, and their regulation through Toll-like receptor signaling. It was generally though that TLR-mediated recognition of specific structures of invading pathogens initiate innate as well as adaptive immune responses through dendritic cells. New evidence suggests that TLR signaling may directly regulate the suppressive function of Treg cells. Linking TLR signaling to the functional control of Treg cells opens intriguing opportunities to shift the balance between CD4(+) T-helper and Treg cells, in ways that may improve the outcome of cancer immunotherapy.