Abstract Ly-6A is a GPI-anchored cell surface protein with reported cell adhesion and cell signaling role in CD4 +T cells. Ly-6A shows low expression on naïve T cells; however, its expression is elevated upon T cell activation. Additionally, IFN-γ and IL-27 cytokines increase Ly-6A expression, a feature observed in immune checkpoint inhibitory (ICI) proteins, such as PD-1 and CTLA-4. Many reports using cell lines and primary cells show, as for ICI proteins, Ly-6A expression inhibiting clonal expansion of antigen receptor-stimulated CD4 +T cells. While the published in vitro and ex vivo studies provide strong evidence for Ly-6A as T cell inhibitory protein, in vivo role of Ly-6A is unknown. Using a tumor transplantation mouse model, we have found that Ly-6A-deficient mice support slower or no growth of B16-F10 melanoma and MC38 adenocarcinoma tumors compared to the wildtype C57BL/6 control mice. We report that Ly-6A-deficient mice injected with B16-F10 tumor cells have elevated number of activated T cells in the draining lymph nodes (DLN) on day-3 post-injection. Furthermore, significantly elevated proliferation of DLN cells from B16-F10 injected Ly-6A-deficient mice was observed on day-7, as assessed by ex vivo proliferation assays. Transcriptome analyses of the tumor tissues showed, among others, significantly elevated expression of IFN-γ inducible and chemokine genes. Additionally, CD73 expression, as assessed by confocal microscopy, was lower in B16-F10 tumor tissues from Ly-6A-deficient mice than the wildtype tumors. Our results reveal a novel immune inhibitory role of Ly-6A protein and potential ICI target for cancer immunotherapy, either as a standalone or combinatorial ICI blockade strategy. Supported by funds from Department of Biology, Villanova University (SRG) & Sigma Xi