Immune checkpoint inhibitors have revolutionized cancer therapy; however, a large proportion of patients do not respond well to current checkpoint immunotherapies. CD200R is recognized as an inhibitory receptor on immune cells, while the therapeutic potential and cellular mechanism of targeting CD200R in tumor immunotherapy remain to be studied. Here we found that CD200R was highly expressed on tumor-infiltrating NK and CD8⁺ T cells with exhausted phenotypes. Either genetic ablation or antibody blockade of CD200R could slow tumor growth and prolong survival of tumor-bearing mice by preventing or reversing exhaustion of both NK cells and CD8⁺ T cells. Additionally, combined therapy of CD200R antibody with anti-PD-1/anti-PD-L1 synergistically inhibited tumor growth. Cellular depletion of NK or/and CD8⁺ T cells demonstrated that both cell types contributed to the anti-tumor efficacy of CD200R blockade in tumor-bearing mice. Further, blockade of human CD200R significantly enhanced human NK cell function and inhibited human tumor growth in PBMC-reconstituted xenograft mice. Our results indicate that CD200R is a potential immune checkpoint molecule that can suppress the tumoricidal activities of NK and CD8⁺ T cells, and could thus be exploited as a therapeutic target in the future.