Background: Although video-assisted thoracoscopic surgery (VATS) segmentectomy has become widespread, the advantage of uniportal VATS (U-VATS) segmentectomy over multiportal VATS (M-VATS) remains controversial. The purpose of this study was to verify the safety and usefulness of U-VATS segmentectomy compared with conventional hybrid/multiportal segmentectomy.Methods: Here, we retrospectively reviewed the data from anatomical pulmonary segmentectomy cases in a single institution from March 2010 to March 2021. Patients were divided into the U-VATS and hybrid/ multiportal VATS (H/M-VATS) groups. Perioperative results were compared between the groups after matching for patient background characteristics. In addition, cases of complex segmentectomy were selected from each group and compared in terms of perioperative results.Results: A total of 180 patients underwent pulmonary segmentectomy during the study period at this institution, comprising 57 cases in the U-VATS group and 123 cases in the H/M-VATS group. After matching for age, sex, disease, tumor location, and type of segmentectomy, no significant differences between the groups were seen in blood loss, major intraoperative bleeding, rate of conversion to thoracotomy, postoperative complications, or re-hospitalization within 30 days after discharge. Operation time (141±46 vs.174±45 min, P<0.001), postoperative drainage duration (1.5±1.2 vs. 2.3±1.8 days, P=0.007), and postoperative hospital stay (3.4±2.0 vs. 4.6±2.5 days, P=0.006) were significantly lower in the U-VATS group. Subgroup analysis of the complex segmentectomy cases also revealed that operation time (146±34 vs. 185±47 min, P<0.001), postoperative drainage duration (1.5±1.3 vs. 2.2±1.2 days, P=0.021), and postoperative hospital stay (3.0±1.4 vs. 4.9±2.1 days, P<0.001) were significantly reduced in the U-VATS group.Conclusions: U-VATS segmentectomy appears as safe and feasible as H/M-VATS segmentectomy. An experienced surgeon can make a smooth transition to U-VATS.