This review focuses on the targeting of tumor-infiltrating regulatory T cells (Ti-Tregs) for improved antitumor responses. Ti-Tregs play a crucial role in suppressing the immune response and maintaining the immunosuppressive microenvironment in tumors. The review highlights the multifaceted mechanisms by which Ti-Tregs exert their immunosuppressive effects, including the expression of molecules such as CTLA-4, PD-1, ICOS, LAG-3, TIGIT, and members of the TNF receptor superfamily, as well as the secretion of immunosuppressive cytokines like IL-10, TGF-β, and IL-35. Additionally, Ti-Tregs utilize chemokine receptors to migrate to the tumor microenvironment and adapt their metabolism to survive in hypoxic and glucose-deficient conditions. The review also discusses various strategies for targeting Ti-Tregs, including specific depletion or inhibition of Tregs, reduced recruitment of Tregs, attenuating their immunosuppressive function, and modified treatment strategies based on kinetics and spatial distinction. These strategies include blocking immune checkpoints, targeting chemokine receptors, inhibiting metabolic pathways, and using targeted therapies that exploit the unique characteristics of Ti-Tregs. The review emphasizes the importance of understanding the tumor immune microenvironment and selecting specialized therapeutic methods to effectively target Ti-Tregs and enhance anti-tumor responses. Overall, the review provides a comprehensive overview of the current understanding of Ti-Tregs and the potential of targeting them for improved cancer treatment.This review focuses on the targeting of tumor-infiltrating regulatory T cells (Ti-Tregs) for improved antitumor responses. Ti-Tregs play a crucial role in suppressing the immune response and maintaining the immunosuppressive microenvironment in tumors. The review highlights the multifaceted mechanisms by which Ti-Tregs exert their immunosuppressive effects, including the expression of molecules such as CTLA-4, PD-1, ICOS, LAG-3, TIGIT, and members of the TNF receptor superfamily, as well as the secretion of immunosuppressive cytokines like IL-10, TGF-β, and IL-35. Additionally, Ti-Tregs utilize chemokine receptors to migrate to the tumor microenvironment and adapt their metabolism to survive in hypoxic and glucose-deficient conditions. The review also discusses various strategies for targeting Ti-Tregs, including specific depletion or inhibition of Tregs, reduced recruitment of Tregs, attenuating their immunosuppressive function, and modified treatment strategies based on kinetics and spatial distinction. These strategies include blocking immune checkpoints, targeting chemokine receptors, inhibiting metabolic pathways, and using targeted therapies that exploit the unique characteristics of Ti-Tregs. The review emphasizes the importance of understanding the tumor immune microenvironment and selecting specialized therapeutic methods to effectively target Ti-Tregs and enhance anti-tumor responses. Overall, the review provides a comprehensive overview of the current understanding of Ti-Tregs and the potential of targeting them for improved cancer treatment.