Background: The importance of fluorescence-based optical imaging in various fields of surgery is increasing. This is a prospective cohort study aimed to investigate the feasibility and efficacy of indocyaninegreen fluorescence (ICGF)-based navigation for near-infrared (NIR) thoracoscopic segmentectomy. Methods: ICGF-based video-assisted thoracoscopic surgery (VATS) segmentectomy was performed in 149 patients. Each patient underwent preoperative evaluation by multidetector-row computed tomography (MDCT), which provided three-dimensional simulations of vascular structures, segmental bronchi, and lung tumor. During the procedure, low-dose ICG (0.25 mg/kg) was injected systemically after the target segmental pulmonary arteries and bronchus were divided. Under NIR-thoracoscopic guidance, an ICG fluorescent line was marked by electric scalpel, followed by division of lung parenchyma along the line by electric scalpel or endoscopic staples. Results: An intersegmental line of ICGF was visible in 98% of patients, even with the use of low-dose ICG. Neither ICG-related adverse events nor procedure-related major complications occurred. The 5-year overall (OS) and recurrence-free survival (RFS) rates were 91.8% and 98%, respectively. Localized recurrence at the resected site did not occur in any patient. Conclusions: ICGF-based navigation for NIR VATS segmentectomy for patients with lung cancer is feasible and effective.