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Background and objective To evaluate the clinical experiences on the application of video-assisted mediastinoscopy for 40 patients with mediastinal lesions. Methods Forty patients underwent video-assisted mediastinoscopy through neck-way surgery, including 17 cases with sole mediastinal nodule or mass, of which the pathologic diagnosis was warranted; 18 cases diagnosed as lung cancer with enlarged mediastinal lymph nodes, of which the clinical stage needed to be clarified; and 5 cases of mediastinal mass with miscellaneous clinical conditions. The results of mediastinoscopy biopsy and pathological diagnosis were analyzed. Results Satisfied tissue for detailed examination was obtained for all of the 40 cases who underwent mediastinoscopy. The successful rate for biopsy was 100%. The final pathologic diagnosis were 11 cases of lymph node metastasis from lung cancer, 4 cases of lymph node metastasis from unknown site, 9 cases of sarcoidosis, 6 cases of lymph node reactive hyperplasia, 4 cases of tuberculosis, 2 cases of lymphoma, 2 cases of mediastinal cyst, 1 case of retrosternal thyroid, 1 case of inflammatory granuloma. Two cases of postoperative hemorrhage related to surgery were successfully treated. Conclusion Video-assisted mediastinoscopy is safe and useful. It's essential for the diagnosis of sole mediastinal mass, a good method to clarify the pathologic stage of patients with lung cancer, and the only way to get the pathologic diagnosis for some special mediastinal disease.