ObjectiveTo summarize the clinical experience of the uniportal thoracoscopic anatomical sub-segmentectomy of the basal segment. MethodsThe clinical data of 34 patients who underwent uniportal thoracoscopic anatomical sub-segmentectomy of the basal segment in our department between April 2018 and April 2021 were retrospectively analyzed. There were 19 males and 15 females with a median age of 56.5 (28.0-76.0) years, a 3-4 cm incision was made in the 5th intercostal area at the front axillary line, and anatomical sub-segmentectomy of the basal segment was performed. Results The surgery was successfully performed in all patients, and there was no patient with additional chest incision or transfer to thoracotomy. The median operation time was 165.0 (125.0-220.0) min, intraoperative blood loss was 120.0 (70.0-290.0) mL, thoracic drainage time was 3.5 (2.0-24.0) d, and hospitalization time was 6.0 (3.0-26.0) d. There was no death during the hospitalization. Postoperative complications included 4 patients of atrial fibrillation, 2 patients of blood sputum, 3 patients of persistent air leakage, and they were recovered after conservative treatment. One patient developed pneumothorax after discharge, 1 patient developed pleural effusion, and both of them recovered after drainage. Postoperative pathology showed microinvasive adenocarcinoma in 22 patients, adenocarcinoma in situ in 7 patients, benign tumors in 5 patients. The lymph nodes were negative in all patients. Conclusion The uniportal thoracoscopic anatomical sub-segmentectomy of the basal segment is safe and feasible, and can be popularized and applied in clinic.