Because of their small size, unique physics, and chemical properties, metal nanoparticles can easily cross obstacles and reach their target cells, which makes them an ideal choice for therapeutic purposes in various cancers. In this study, the effects of iron oxide nanoparticles on MDA-MB-231 breast cancer cell line were examined, and biomarkers related to oxidative stress were evaluated. Fe2O3 nanoparticles were suspended in a cell culture medium and diluted to appropriate concentrations (0, 10, 30, 60, and 120 μg/ml) for 24 and 48 h. GSH, superoxide dismutase, catalase, and ROS generation were evaluated. The results showed that iron oxide nanoparticles induced intracellular ROS generation in a dose- and time-dependent manner. The results further showed that iron oxide nanoparticles increased ROS and activated oxidative stress in cells.