Regulatory T cells (Tregs) are abundant in tumor tissues, raising a question of whether immunosuppressive tumor-infiltrating Tregs (TI-Tregs) can be selectively depleted or functionally attenuated to evoke effective anti-tumor immune responses by conventional T cells (Tconvs), without perturbing Treg-dependent immune homeostasis in healthy organs and causing autoimmunity. Here, we review current cancer immunotherapy strategies, including immune checkpoint blockade (ICB) antibodies against CTLA-4 and PD-1 and discuss their effects on TI-Tregs. We also discuss approaches that exploit differentially regulated molecules on the cell surface (e.g., CTLA-4) and intracellularly (e.g., T cell receptor signaling molecules) between TI-Tregs and Tconvs as well as their dependence on cytokines (e.g., IL-2) and metabolites (e.g., lactate). We envisage that targeting TI-Tregs could be effective as a monotherapy and/or when combined with ICB antibodies.