Resistance to anti–PD-1/PD-L1 treatment is often associated with accumulation of intratumoral inhibitory macrophages. V-domain immunoglobulin suppressor of T-cell activation (VISTA) is a nonredundant immune checkpoint that can induce both T-cell and myeloid-cell immunosuppression. In this study, we found that high levels of VISTA+ immune cells were associated with advanced stage bladder cancer and predicted poor survival in patients. A combination of high infiltration of VISTA+ immune cells and PD-L1+ immune cells or PD-1+ T cells predicted the worst survival. Flow cytometry and multiplex immunofluorescence analyses confirmed that VISTA expression was higher in macrophages than in T cells or neutrophils, and only VISTA+CD163+ macrophage density predicted poor prognosis in patients with bladder cancer. Toll-like receptor (TLR) agonists are known to trigger the innate immune response in macrophages. We found that the VISTA-specific mAb 13F3 augmented the ability of a TLR3-specific adjuvant to induce macrophage activation in vitro. In the MB49 syngeneic mouse model of bladder cancer, treatment with 13F3 curbed tumor growth and prolonged survival when combined with a TLR3-specific adjuvant. The combination treatment reduced the intratumoral frequency of CD206+ anti-inflammatory macrophages and levels of the immunosuppressive molecule TGFβ1, but it upregulated expression of immunostimulatory molecules (Ifna, Ifnb, and Trail) and increased the CD8+ T cell/regulatory T-cell ratio. These findings indicate that elevated VISTA expression in immune cells, particularly macrophages, is associated with an unfavorable prognosis in patients with bladder cancer and suggest that targeting VISTA in combination with a TLR3-specific adjuvant has translational potential.