Indocyanine green (ICG), an organic anion used in liver function tests, is known to accumulate in hepatocellular carcinoma (HCC) tissues after an intravenous injection. Because the intratumoral expression of transporters for chemical agents influences the behaviors of some malignant tumors, we investigated whether the expression of ICG-related transporters influenced the clinicopathologic features of HCC.ICG accumulation patterns were examined using near-infrared spectroscopy and the protein expression of ICG-related transporters was assessed using immunohistochemistry and immunoblotting in 40 resected HCC specimens. We also determined whether the intratumor expression of these transporters affected intratumor lipid composition by imaging mass spectrometry.Immunoblot analysis revealed that the expression of organic anion transporting polypeptide 1B3 (OATP1B3) and multidrug resistance p-glycoprotein (MDR)-3, as influx and efflux transporters, respectively, were significantly higher in ICG-accumulated HCC (ICG-high HCC) than in ICG-low HCC. ICG was fluorescently observed in the pseudoglands and bile canaliculi abundantly expressing MDR3. An immunohistochemical examination revealed significantly worse disease-free and overall survival rates in patients with MDR3-negative HCC, in which the intratumoral accumulation of some phosphatidylcholine species was observed under imaging mass spectrometry.The intratumoral expression of MDR3, a key efflux transporter of ICG, affected the prognosis of patients with HCC, presumably by altering the lipid composition of the lipid bilayers.