Abstract: Laparoscopic fenestration (LF) has recently been considered a standard procedure for nonparasitic symptomatic liver cysts. Here, we report a case of LF that was safely performed using real-time indocyanine green (ICG) fluorescence-guided surgery. A 74-year-old woman presented with right upper abdominal pain and poor dietary intake. The patient was diagnosed with symptomatic liver cysts and underwent LF. One hour before surgery, ICG (2.5 mg) was intravenously administered to the patient. ICG fluorescence imaging clearly showed the biliary ducts and distinguished the cysts from the liver parenchyma. We could resect only the cyst walls as wide as possible under the guidance of both white light and fluorescence imaging. There were no signs of postoperative symptom recurrence. Detection of ICG fluorescence in the liver parenchyma is as important as ICG cholangiography for fenestration. Laparoscopic liver cyst fenestration with real-time ICG fluorescence-guided surgery is safe and can be used as a standard procedure.