Objective To investigate the effect of modular disscection of mediastinal lymphadenectomy in uniportal video-assisted thoracoscopic surgery (uniportal-VATS) for lung cancer radical resection and assess its safety and feasibility. Methods Data of 311 patients (171 males and 140 females, a mean age of 59.4±5.1 years) with non-small cell lung cancer (NSCLC) who received modular dissection of mediastinal lymphadenectomy in uniportal-VATS or three portal VATS (3P-VATS group) during March to December 2015 were retrospectively analyzed. There were 208 patients (110 males and 98 females, a mean age of 59.2±5.3 years) in the uniportal-VATS group and 103 patients (61 males and 42 femals, a mean age of 59.7±5.1 years) in the 3P-VATS group. The effects of lymph nodes (LNs) dissection and postoperative clinical data were compared between the two groups, especially for N2 LNs dissection. Results There were no perioperative death in two groups. The overall number of dissected stations and LNs in the uniportal-VATS group (7.3±1.0, 17.5±3.0) were similar with those in the 3P-VATS group (7.2±1.0, 17.7±2.7, P=0.208, P=0.596). The dissected stations (uniportal-VATS: 4.3±0.7, 3P-VATS: 4.3±0.6) and number (uniportal-VATS: 8.6±1.1, 3P-VATS: 8.5±1.1) of N2 LNs were both similar between the two groups (P=0.850, P=0.587). The chest tube duration and postoperative hospital stay of uniportal-VATS group (4.4±1.3 d and 9.2±0.9 d) were much shorter than those of 3P-VATS group (5.0±1.3 d and 9.8±2.0 d, both P<0.001). No significant difference was found in morbidity rate between the two groups (P>0.05). Conclusion Modular dissection of mediastinal lymphadenectomy could meet the requirment of radical resection and it is a safe and valid method which could be used during uniportal-VATS for radical resection of lung cancer.