The core purpose of cancer immunotherapy is the sustained activation and expansion of the tumor specific T cells, especially tumor-infiltrating cytotoxic T lymphocytes (CTLs). Currently, one of the main foci of immunotherapy involving nano-sized carriers is on cancer vaccines and the role of professional antigen presenting cells, such as dendritic cells (DCs) and other phagocytic immune cells. Besides the idea that cancer vaccines promote T cell immune responses, targeting immune inhibitory pathways with nanoparticle delivered regulatory agents such as small interfering RNA (siRNA) to the difficultly-transfected tumor-infiltrating T cells may provide more information on the utility of nanoparticle-mediated cancer immunotherapy. In this study, we constructed nanoparticles to deliver cytotoxic T lymphocyte-associated molecule-4 (CTLA-4)-siRNA (NPsiCTLA-4) and showed the ability of this siRNA delivery system to enter T cells both in vitro and in vivo. Furthermore, T cell activation and proliferation were enhanced after NPsiCTLA-4 treatment in vitro. The ability of direct regulation of T cells of this CTLA-4 delivery system was assessed in a mouse model bearing B16 melanoma. Our results demonstrated that this nanoparticle delivery system was able to deliver CTLA-4-siRNA into both CD4+ and CD8+ T cell subsets at tumor sites and significantly increased the percentage of anti-tumor CD8+ T cells, while it decreased the ratio of inhibitory T regulatory cells (Tregs) among tumor infiltrating lymphocytes (TILs), resulting in augmented activation and anti-tumor immune responses of the tumor-infiltrating T cells. These data support the use of potent nanoparticle-based cancer immunotherapy for melanoma. T cell mediated immunotherapy is an effective treatment option for malignant melanoma. It is critical for such immunotherapy to obtain a sufficient number of functional/activated T cells. However, CTLA-4 plays a potent inhibitory role in T cell activation and proliferation, which significantly curbs T cell-mediated tumor rejection. Hence, we investigated a method to exploit a nanoparticle delivery system to efficiently deliver siRNA (NPsiCTLA-4) targeting an immune checkpoint molecule, i.e. cytotoxic T lymphocyte-associated molecule-4, to manipulate or modulate tumor-infiltrating T cells and to assess the effects of NPsiCTLA4 on the blockade of CTLA-4 and the resulting enhancement of T cell mediated anti-tumor immunotherapy. Display omitted