The induced membrane damage is a key mechanism for the cytotoxicity of graphene nanosheets (GNSs). In this research, the physical interaction of GNSs on model membranes was investigated using artificial membranes and plasma membrane vesicles. The effects of the GNSs on plasma membrane, lysosomal and mitochondrial membranes were investigated using rat basophilic leukemia (RBL2H3) cells via lactate dehydrogenase (LDH) assay, acridine orange staining and JC-1 probe, respectively. The physical interaction with model membranes was dominated by electrostatic forces, and the adhered GNSs disrupted the membrane. The degree of physical membrane disruption was quantified by the quartz crystal microbalance with dissipation (QCM-D), confirming the serious membrane disruption. The internalized GNSs were mainly distributed in the lysosomes. They caused plasma membrane leakage, increased the lysosomal membrane permeability (LMP), and depolarized the mitochondrial membrane potential (MMP). The increased cellular levels of reactive oxygen species (ROS) were also detected after GNS exposure. The combination of physical interaction and the excess ROS production damaged the plasma and organelle membranes in living RBL-2H3 cells. The lysosomal and mitochondrial dysfunction, and the oxidative stress further induced cell apoptosis. Specially, the exposure to 25 mg/L GNSs caused severest cell mortality, plasma membrane damage, ROS generation, MMP depolarization and apoptosis. The research findings provide more comprehensive information on the graphene-induced plasma and organelle membrane damage, which is important to understand and predict the cytotoxicity of carbon-based nanomaterials.Copyright © 2020 Elsevier B.V. All rights reserved.