Liver dysfunction affects a variety of metabolic pathways in the critically ill, but mechanisms remain poorly understood. We prospectively assessed markers of hepatic injury and function in sepsis and I/R injury in vivo and molecular mechanisms in human liver tissue ex vivo. Markers of hepatocellular injury, synthesis, and excretion, including plasma disappearance rate of indocyanine green (ICG), were measured in 48 patients with severe sepsis. Incidence of liver dysfunction was 42% as assessed by hyperbilirubinemia but 74% by impaired dye excretion. Conventional markers for liver injury failed to predict outcome, whereas dye excretion of less than 8% per minute predicted death with high sensitivity and specificity. Potential mechanisms were assessed via (a) gene expression analysis of transporter proteins for bilirubin and ICG in cultured human liver tissue, and (b) monitoring uptake and excretion of the dye after I/R injury in 12 patients receiving a biliary T-tube during liver transplantation. Ex vivo gene expression of transporters was differentially affected for bilirubin and ICG with upregulation of basolateral and downregulation of canalicular ICG transporters. Consistently, patients with unfavorable course after liver transplantation displayed almost complete cessation of biliary dye excretion, whereas uptake into the hepatocyte was reduced by only 40%. In conclusion, standard liver tests lack the required sensitivity to assess hepatic injury and function in the critically ill. Dye excretion better reflects excretory and/or microvascular dysfunction but still underestimates impaired canalicular transport. The observed differential susceptibility of the polar surfaces of human hepatocytes has potential implications for monitoring liver function and drug-induced liver injury.