Background Near-infrared indocyanine green fluorescence cholangiography (NIRF) has shown promising results on delineating extra-hepatic biliary anatomy during laparoscopic cholecystectomy to avoid bile duct injury. However its routine usage remains in question. In this study, the technique was evaluated further with learning curve estimation and learning factors were observed. Methods One hundred ninety-nine cases which underwent laparoscopic cholecystectomy for acute or chronic cholecystitis within a 2-year period including 51 cases with initial use of NIRF by 2 surgeons were studied retrospectively. The learning curve was evaluated for a surgeon as primary objective. A case-matched comparison of the operative time between NIRF and conventional group, in terms of acute and chronic cholecystitis was also conducted as a secondary calculation. Results Learning curve was evaluated with 61% learning rate for NIRF experience. Cysto-biliary junction non-illuminated cases under fluorescent view, had mean operative time of 80.83 ± 22.82 min, which was shorter than the cysto-biliary junction illuminated cases. The NIRF group exhibited longer operative time compared with the conventional group with mean difference of 34.39 min (significant at P < .05). Conclusions While the initial learning phase might be affected by surgeons’ behavior and attitude, our results may provide a reference to learn at one’s own pace and to employ NIRF teaching strategies during surgical training programs to overcome the initial phase during training period itself and facilitate universal achievement.