Perioperative imaging with indocyanine green (ICG) is developing to increase safety in dissecting anatomical structures during hepatobiliary surgery. Images obtained with the fluorescence camera rely on concentrations measured in liver regions of interest. However, how ICG sinusoidal uptake and hepatocyte elimination rates generate ICG hepatocyte concentrations is largely unknown. To investigate such issue and better understand the role of membrane transporters in generating ICG hepatocyte concentrations, we perfused ICG in livers isolated from normal livers. Whether the well‐known transporter inhibitor rifampicin modifies hepatocyte ICG concentrations was also studied. The dye has a very high and constant extraction ratio (96%) into hepatocytes. This persistent high extraction ratio generates a huge uphill concentration gradient across the sinusoidal membrane: from 5 μM (sinusoids) to 1600 μM (liver). When inside hepatocytes, ICG has low hepatocyte elimination (7 nmol/min.) and liver concentrations do not decrease much over time. Moreover, the tiny hepatocyte ICG efflux is mainly due to ICG return back to sinusoids (90%). Rifampicin slightly inhibits ICG uptake into hepatocytes and when inside hepatocytes blocks ICG efflux into bile canaliculi. In contrast, it increases ICG efflux back to sinusoids with significant decrease in ICG liver concentrations. Imaging with ICG in the perioperative period reflects the high hepatocyte concentrations and relies on the high extraction ratio across hepatocyte sinusoidal membrane. Although ICG concentrations are low in bile ducts, they are adequate for a good visualization and avoid bile duct injury.