The immunoregulatory protein T-cell immunoglobulin- and mucin-domain–containing molecule-3 (Tim-3) mediates T-cell exhaustion and contributes to the suppression of immune responses in both viral infections and tumors. Tim-3 blockade reverses the exhausted phenotype of CD4⁺ and CD8⁺ T cells in several chronic diseases, including melanoma. Interestingly, natural killer (NK) cells constitutively express Tim-3; however, the role of Tim-3 in modulating the function of these innate effector cells remains unclear, particularly in human diseases. In this study, we compared the function of Tim-3 in NK cells from healthy donors and patients with metastatic melanoma. NK cells from the latter were functionally impaired/exhausted, and Tim-3 blockade reversed this exhausted phenotype. Moreover, Tim-3 expression levels were correlated with the stage of the disease and poor prognostic factors. These data indicate that Tim-3 can function as an NK-cell exhaustion marker in advanced melanoma and support the development of Tim-3–targeted therapies to restore antitumor immunity. Cancer Immunol Res; 2(5); 410–22. ©2014 AACR.