Regulatory T cells (Treg cells) have reshaped modern immunology by establishing the conceptual and mechanistic foundation of peripheral immune tolerance. Since the pioneering identification of CD4?CD25? suppressive T cells by Shimon Sakaguchi and the subsequent discovery of the lineage-defining transcription factor Forkhead box P3 (Foxp3) by Mary E. Brunkow and Fred Ramsdell, Treg cells have been recognized as indispensable guardians of immune homeostasis. These advances collectively clarified that central tolerance alone is insufficient to eliminate all self-reactive lymphocytes, and peripheral tolerance—critically mediated by Treg cells—serves as a second barrier preventing pathological autoimmunity. Contemporary research has therefore expanded the functional and therapeutic significance of Treg cells across the fields of autoimmunity, cancer, transplantation, and tissue repair. Treg cells originate from two major developmental pathways: thymus-derived Treg (tTreg) cells, which arise from high-affinity self-reactive TCR interactions in the thymus, and peripheral Treg (pTreg) cells, which are induced in mucosal and other peripheral tissues via antigen stimulation under tolerogenic cytokine cues such as IL-2 and TGF-β. Their differentiation is orchestrated by a multilayered transcriptional and epigenetic network within the Foxp3 locus, including CNS0–CNS3 elements that integrate TCR, cytokine and environmental signals to support lineage stability. Treg cells are identified by a combination of surface and intracellular markers—CD25, CD127low/-, CTLA-4, GITR, TNFR2, CD39/CD73, and Foxp3—although marker specificity varies with context, activation state, and species. Their notable heterogeneity enables Treg cells to adopt Th1-, Th2-, Th17- or Tfh-like programs through transcription factors such as T-bet, GATA3, RORγt and Bcl6, thereby permitting precise suppression of corresponding effector responses. Tissue-resident Treg subsets in adipose tissue, skin, skeletal muscle and the CNS have emerged as highly specialized regulators that integrate local metabolic and stromal signals, contributing not only to immunosuppression but also to tissue regeneration. Mechanistically, Treg cells maintain tolerance through three synergistic strategies: (1) secretion of suppressive cytokines (IL-10, TGF-β, IL-35) and cytotoxic mediators (granzyme B, perforin); (2) cell-contact–dependent interactions via CTLA-4, PD-1/PD-L1, and LAG-3 to limit dendritic cell maturation and T-cell activation; and (3) metabolic regulation including IL-2 consumption, adenosine production via CD39/CD73, cAMP transfer through gap junctions, and adaptation to hypoxic or nutrient-restricted microenvironments. Dysregulation of Treg cell quantity or function contributes directly to pathogenesis across a spectrum of diseases. In autoimmune diseases such as type 1 diabetes, systemic lupus erythematosus, rheumatoid arthritis and multiple sclerosis, impaired Foxp3 stability, epigenetic abnormalities, defective IL-2 signaling or inflammatory cytokine exposure undermine Treg suppressive capacity, facilitating excessive autoreactive T- and B-cell activation. In contrast, within the tumor microenvironment, Treg cells are often enriched through chemokine axes such as CCL22–CCR4 and reinforced by interaction with myeloid-derived suppressor cells and tumor-associated macrophages. Their enhanced metabolic fitness and suppressive phenotype enable tumors to evade immune destruction. In transplantation, Treg cells are essential for promoting graft tolerance, restraining effector T-cell activation, and facilitating tissue repair after injury. Rapid therapeutic progress has been driven by Treg-based immunomodulation. Polyclonal Treg adoptive transfer has demonstrated safety and preliminary efficacy in type 1 diabetes, autoimmune disorders, solid-organ transplantation, and graft-versus-host disease. Gene-engineered Treg therapies, including antigen-specific CAR-Treg and TCR-Treg platforms, offer superior precision and stability, enabling targeted suppression at disease sites. Additional strategies—including low-dose IL-2 therapy, small-molecule modulation, and selective depletion of intratumoral Treg using antibodies against CCR4, CCR8, CTLA-4 or CD25×TIGIT bispecifics—further expand the translational landscape. Collectively, advances in Treg biology—from lineage ontogeny and molecular regulation to specialized functions and therapeutic engineering—highlight Treg cells as central orchestrators of immune equilibrium. Continued integration of single-cell multi-omics, systems immunology and gene-editing technologies is expected to accelerate the development of highly specific, durable and safe Treg-centered therapies, ultimately enabling precision control of immune tolerance in autoimmunity, transplantation and cancer.