Magnetic nanoparticles (NPs) are used to a large extent in the targeted delivery of therapeutic agents. In this study, we aimed to investigate the possible toxicity of Fe2O
3 NPs on human cells, including blood lymphocytes. We isolated blood lymphocytes from healthy humans using Ficoll polysaccharide and subsequently by gradient centrifugation. Then, the toxicity parameters, including cell viability, reactive oxygen species (ROS) formation, lipid peroxidation, cellular glutathione (GSH) level, mitochondrial and lysosomal damage, were measured in blood lymphocytes after exposure to Fe
2O
3 NPs. Our results indicated that Fe
2O
3 NPs significantly (dependent on concentration) reduced the cell viability, and the IC
50 was determined to be 1 mM. With increasing concentrations, we found that Fe
2O
3 NPs–induced cell toxicity was associated with a significant increase in intracellular ROS and loss of mitochondrial membrane potential and lysosomal membrane leakiness. Consequently, these NPs at different concentrations affect GSH level and cause oxidative stress in human lymphocytes.