Tumor-infiltrating regulatory T cells (TI-Tregs) are characterized by their abnormal accumulation and heightened immunosuppressive activity. However, the biomechanical mechanisms that govern Treg identity and function through extracellular matrix (ECM) properties remain poorly understood. In three-dimensional culture systems and the tumor microenvironment (TME), increased matrix stiffness and viscoelasticity have been shown to promote Treg differentiation and expansion. Structural remodeling of the ECM, particularly the realignment of collagen fibers and the reduction in effective pore size, significantly enhances Treg migration. Moreover, biomechanical signals derived from the ECM strengthen the oxidative phosphorylation (OXPHOS) metabolic phenotype and immunosuppressive function of Tregs by modulating mitochondrial dynamics. This review provides a comprehensive analysis of the molecular events through which ECM mechanical properties-such as stiffness, viscoelasticity, and topological structure-regulate Treg identity and functionality, as well as the mechanical sensing and response mechanisms employed by Tregs. The potential for targeting Treg mechanosensors and mechanotransduction pathways to develop mechano-immunomodulatory strategies for cancer therapy is also discussed.© 2025. The Author(s).