V-domain Ig suppressor of T cell activation (VISTA) is a novel negative checkpoint ligand that is homologous to PD-L1 and suppresses T cell activation. This study demonstrates the multiple mechanisms whereby VISTA relieves negative regulation by hematopoietic cells and enhances protective anti-tumor immunity. VISTA is highly expressed on myeloid cells and Foxp3+CD4+ regulatory cells, but not on tumor cells within the tumor microenvironment (TME). VISTA monoclonal antibody (mab) treatment increased the number of tumor-specific T cells in the periphery, and enhanced the infiltration, proliferation and effector function of tumor-reactive T cells within the TME. VISTA blockade altered the suppressive feature of the TME, by decreasing the presence of monocytic myeloid-derived suppressor cells and increasing the presence of activated DCs within the TME. In addition, VISTA blockade impaired the suppressive function and reduced the emergence of tumor-specific Foxp3+CD4+ regulatory T cells. Consequently, VISTA mab administration as a monotherapy significantly suppressed the growth of both transplantable and inducible melanoma. Initial studies explored a combinatorial regimen using VISTA blockade and a peptide-based cancer vaccine with TLR agonists as adjuvants. VISTA blockade synergized with the vaccine to effectively impair the growth of established tumors. Our study therefore establishes a foundation for designing VISTA-targeted approaches either as a monotherapy or in combination with additional immune-targeted strategies for cancer immunotherapy.