Near infrared (NIR) fluorescence-guided pulmonary segmentectomy following endobronchial or intravenous indocyanine green (ICG) administration has been developed and reported. The aim of this study is to prospectively validate the feasibility and safety of NIR fluorescence-guided pulmonary segmentectomy following endobrochial ICG injection using navigational bronchoscopy. Patients who underwent pulmonary segmentectomy were prospectively enrolled in this study. Using preoperative CT datasets a 3D image of target segments was reconstructed for lung volumetry and a bronchial road map was created to determine the bronchus for ICG injection. The ICG concentration was 0.125 mg/mL. Immediately after intubation the ICG was injected into the target bronchi using an ultrathin bronchoscope followed by air flushing to expedite ICG dispersion to the periphery. A NIR thoracoscope (PINPOINT, Novadaq) was used to detect ICG fluorescence and determine intersegmental plane for pulmonary segmentectomy. Usefulness and safety of this technique were evaluated by 1) whether ICG demarcation lines correspond to intersegmental lines expected from pulmonary veins, 2) whether large bronchi and vessels in adjacent segments emerge when dividing intersegmental planes using electrical cautery. The patients were followed up to 1 month after surgery to see if any complication existed. Eight male and 7 female patients with a mean age of 66.5 ± 9.6 years were enrolled. Segmentectomy regions included right S1, S2, S6, S8 and S10 segments, and left S1+2+3, lingular, S6, S8, S9+10, and basilar segments. The average bronchoscopic procedure time was 14.3 ± 8.0 minutes. Vital signs were kept stable before and after the bronchoscopic procedure. The mean injected volume of ICG solution was 21.2 ± 8.8 mL as per a case. In 13 out of 15 cases (86.7%), NIR fluorescence guidance was recognized as effective for pulmonary segmentectomy. Intersegmental plane could not be determined in 2 cases likely due to insufficient air flushing, leading to the failure of ICG dispersion to the periphery. There were no complications developed intraoperatively. The average operation time was 193 ± 41 minutes, with a mean bleeding of 110 ± 101 mL. The average duration of drainage was 3.1 ± 1.0 days. Recurrent air leakage happened on postoperative day 6 in 1 case. Otherwise, no procedure related adverse event was noted. NIR fluorescence-guided pulmonary segmentectomy following endobrochial ICG injection using navigational bronchoscopy appeared to be safe and feasible. Sufficient air flushing may be the key for clear ICG demarcation of referred segments.