Abstract: Two-dimensional (2D) graphene oxide (GO) has been widely used for biomedical applications such as drug delivery, biosensing and bioimaging. However, problems still exist with respect to their toxicity to major organs such as the liver. In this study, we systemically investigated the impact of GO on three major liver cell types: Kupffer cells, liver sinusoidal endothelial cells (LSEC), and hepatocytes. Pristine and reduced GO, nanosheets (pGO and rGO) with two sizes (about 510 nm and 110 nm) were synthesized. These materials elicited different cellular responses depending on the oxidation state as well as lateral size of GO. While pGO induced plasma membrane damage and necrosis (large nanosheets > small nanosheets) in Kupffer cells, minimal cytotoxicity was observed in LSEC and hepatocytes. In contrast, rGO induced apoptosis in Kupffer cells (large nanosheets > small nanosheets) and LSEC, with negligible effects in hepatocytes. While pGO could attach to the Kupffer cell membrane and induce lipid peroxidation and cytoskeleton disruption, rGO was mostly taken up in Kupffer cells. Moreover, while pGO and rGO were taken up in roughly similar amounts by LSEC, little was internalized for hepatocytes. Studies on the intracellular effects of the GO species at the lysosomal and inflammasomal level demonstrated that rGO damaged lysosomes and induced IL-1β production in both Kupffer and LSEC cells, while similar effects were absent in hepatocytes or with pGO. Our research reveals that the surface oxidation state and lateral size of GO determine their biological effect on tuning the fate of liver cells.