Objective To discuss the pathological diagnostic process of one case of invasive adenocarcinoma of lung complicated with metastatic nuclear protein of testis （NUT） midline carcinoma of mediastinal lymph node，and to provide the basis for the clinical diagnosis of this disease. Methods The clinical materials of one patient with invasive adenocarcinoma of lung complicated with metastatic NUT midline carcinoma of mediastinal lymph node were collected. Intraoperative frozen section pathological diagnosis of the lung tumor was performed to determine the nature of the lesion， and postoperative mediastinal tumor was sent for slow pathological examination. Both lung and mediastinal tumors underwent routine pathological examination and immunohistochemical staining， the pathological diagnostic process was analyzed combined with the relevant literatures， and the pathological differentiation was performed. Results The patient， a 59-year-old man， underwent a CT scan at an external hospital， which revealed a soft tissue density shadow in the anterior mediastinum and a ground-glass nodule in the lower lobe of the right lung， both were considered to be neoplastic lesions. The intraoperative rapid pathological diagnosis of the lung tumor suggested pulmonary invasive adenocarcinoma， and the postoperative immunohistochemical staining results confirmed it as primary pulmonary invasive adenocarcinoma. The postoperative mediastinal tumor was confirmed as lymph node metastatic NUT midline carcinoma through immunohistochemical staining， external consultation， and genetic testing. Conclusion NUT midline carcinoma is a rare poorly differentiated squamous cell carcinoma that often occurs in the midline structures and may involve other organs and lymph node metastasis； its diagnosis requires a combination of histological morphology， imaging data， and genetic testing results.