Background This study is to evaluate the feasibility and safety of video-assisted thoracoscopic (VATS) lobectomy with two incisions. Methods A total of 73 patients (male 47, female 26; mean age 61.2 ± 12.00 years old) who underwent major pulmonary resection, through VATS, using two incisions were included in this study. The thoracoscopy port was placed at the 7th or the 8th intercostal space in the mid-axillary line, and the working port, 3~5 cm long, at the 5th intercostal space, on the operator’s side. Results The preoperative diagnosis was benign lung disease in 8 patients (11.0%) and malignant lung disease in 65 (89.0%). Two patients (3.1%) needed a third port during surgery due to severe pleural adhesion, and conversion to thoracotomy was needed in 5 (6.8%), due to bleeding at pulmonary arterial branch (n = 3), anthracofibrotic lymph nodes around pulmonary artery (n = 1), and severe pleural adhesion (n = 1). The mean duration of the operation in the 66 patients, completed by a two-incision VATS lobectomy, was 163.4 ± 30.40 minutes. In 56 cases, which were completed by a two-incision VATS lobectomy for primary lung cancer, a total number of dissected lymph nodes per patient were 20.2 ± 11.2. The chest tube was removed on postoperative day 5.4 ± 2.8, and there was no occurrence of major perioperative morbidity and mortality. Conclusions Two-incision VATS lobectomy is applicable in the selected cases, and may obtain similar results with the conventional VATS lobectomy, through a certain period of learning curve.