The therapeutic options of non-small cell lung cancer (NSCLC) are limited, although a combination of targeted therapy and immunotherapy is promising. To explore novel targets for immunotherapy, we explored the role of Ccr4-Not transcription complex subunit 4 (CNOT4) in NSCLC. The expression of CNOT4 in tumor tissues was determined by immunohistochemistry staining and western blotting. The cell lines that stably express CNOT4 were established in H1299 and A549 cells. Direct cell counting, MTT assay, and colony formation were used to determine the ability of cell proliferation. Cell apoptosis and cell cycle were next analyzed by PI/Annexin V staining. Cell invasion and migration were examined by transwell assays. To further explore the function of CNOT4 in cytotoxic T lymphocytes (CTLs) mediated cytotoxicity, an in vitro co-culture system of CNOT4 overexpressing and control H1299 cells with CTLs was developed. CNOT4 was down-regulated in tumor tissues compared with paired normal tissues from patients with lung cancers. CNOT4 overexpression significantly inhibited tumor cell proliferation, colony formation, cell migration, and invasion, but promoted cell apoptosis. Furthermore, overexpression of CNOT4 enhanced cytotoxicity of CTLs to H1299. CNOT4 functions as a potential tumor suppressor of NSCLC via inhibiting tumor cell function and increasing the sensitivity to CTLs.