Abstract: Regulatory T cells (Tregs) are the most important population of immune cells in maintaining peripheral immune tolerance in the body. Targeting Tregs to rebuild peripheral immune tolerance has shown broad potential for application in the treatment of various inflammatory diseases, such as autoimmune diseases, allergic disorders, graft-versus-host disease, and organ transplant rejection. However, substantial challenges remain in translating Treg-based therapies into clinical practice. In this review, we first summarize the discovery of Tregs and the principal mechanisms through which Tregs inhibit immune responses. Then, the research progress in polyclonal Treg-based therapy, antigen-specific Treg-based therapy, and low-dose interleukin-2 (IL-2) therapy and the implementation status of clinical trials of Treg therapies were comprehensively summarized. Finally, four issues, including maintaining the stability of Tregs, preparing autoantigen-specific Tregs, such as chimeric antigen receptor (CAR)-Tregs and T-cell receptor (TCR)-Tregs, reshaping the inflammatory microenvironment of diseases, and minimizing the potential adverse effects of Treg therapies, were highlighted as the bottleneck problems in the clinical translation of Treg therapies and priority directions for future research.