Iron oxide nanoparticles (IONPs) with diameters of 15, 25, and 41 nm were evaluated as mediators of thermal cytotoxicity under radio-frequency (RF) exposure. The 25 nm IONPs were found to be the most efficient of the three in killing cancer cells at 350 kHz low-frequency RF irradiation. However, at a higher frequency of 13.56 MHz, 15 nm IONPs produced the highest percentage of cell death. Moreover, the killing effect was concentration-dependent in that a higher concentration of IONPs resulted in increased cellular death. Size-dependent internalization of IONPs in MCF-7 cells was quantified by using inductively coupled-plasma mass spectrometry (ICP-MS). Dark-field microscopy and transmission electron microscopy (TEM) revealed that MCF-7 cells internalize IONPs through endocytosis after 24 hours of incubation. In addition, after RF treatment, the cancer cells underwent the apoptosis process, and the level of reactive oxygen species (ROS) increased significantly after hyperthermia. Scanning electron microscopy (SEM) and TEM further established that the ultrastructure morphological changes in the cancer cells originated from the apoptosis process.