T cell immunoglobulin- and mucin-domain-containing molecule-3 (Tim-3) has been shown to play an important role in mediating NK-cell function in human diseases. However, the relationship between Tim-3 expression in natural killer (NK) cells and human lung adenocarcinoma remains unclear. We therefore investigated the expression of Tim-3 in NK cells and explored the effect of Tim-3 blockade on NK cell-mediated activity in human lung adenocarcinoma. Upregulated expression of Tim-3 on CD3-CD56 + cells (P < 0.05) and CD3-CD56^dim cells (P < 0.05) of patients with lung adenocarcinoma was detected by flow cytometry. Moreover, Tim-3 expression in CD3-CD56 + NK cells was higher in patients with lung adenocarcinoma with lymph node metastasis (LNM) (P < 0.05) or with tumor stage T3–T4 (P < 0.05). Tim-3 expression in CD56^dim NK-cell subset was higher in patients with tumor size ≥ 3 cm (P < 0.05), or LNM (P < 0.05) or with tumor stage T3–T4 (P < 0.05). Further analysis showed that higher expressions of Tim-3 on both CD3-CD56 + NK cells and CD56^dim NK-cell subset were independently correlated with shorter overall survival of patients with lung adenocarcinoma (log-rank test, P = 0.0418, 0.0406, respectively). Importantly, blockade of Tim-3 signaling with anti-Tim-3 antibodies resulted in the increased cytotoxicity and IFN-γ production of peripheral NK cells from patients with lung adenocarcinoma. Our data indicate that Tim-3 expression in NK cells can function as a prognostic biomarker in human lung adenocarcinoma and support that Tim-3 could be a new target for an immunotherapeutic strategy.