Background: The optimal indocyanine green (ICG) administration protocol for fluorescence cholangiography during laparoscopic cholecystectomy (LC) has yet to be determined. Methods: A prospective study including 20 cases of ICG fluorescence-navigated LC was conducted. Accordingly, the first 10 patients were administered 2.5 mg of ICG on the day of surgery after intubation (surgery-day group), while the remaining 10 consecutive patients were administered 0.25 mg/kg of ICG on the evening before surgery (one-day-before group). Fluorescence intensity (FI) of each tissue and FI ratios were then compared between both groups. Results: The median interval between observation and ICG administration was 27 minutes and 16 hours 24 minutes in the surgery-day and one-day-before group, respectively. Although FI values for the common bile duct (CBD), liver, and hepatoduodenal ligament (HDL) were significantly lower in the one-day-before group than in the surgery-day group, CBD-Liver contrast (0.7, 0.6-1.2 vs 2.5, 0.9 =-4.8; P < .001), and CBD-HDL contrast (1.7, 1.4-2.4 vs 2.3, 1.5-13.3; P = .038) were significantly higher in the one-day-before group than in the surgery-day group. Conclusion: ICG administration a day before LC may offer better CBD background contrast compared to administration just prior to surgery.