Iron oxide nanoparticles (IONPs) have shown great potential in various biomedical applications. However, information on the interaction between IONPs and biological systems, especially the uptake and distribution of IONPs in cells and tissues, as well as the mechanism of biological action, is relatively limited. In the present study, multi-modal visualization methods, including confocal fluorescence microscopy, transmission electron microscopy, magnetic resonance imaging, and fluorescence optical imaging, were utilized to unveil the uptake and distribution of IONPs in macrophages, cancer cells, and xenograft models. Our results demonstrated that uptake of IONPs in RAW264.7 macrophages and SKOV-3 cancer cells were dose- and cell type-dependent. Cellular uptake of IONPs was an energy-dependent process, and caveolae-mediated endocytosis was the main uptake pathway. All the IONPs were primarily present in endocytic compartments (e.g., endosomes, lysosomes) inside the cells. At 48 hours after intravenous injection of IONPs in SKOV-3 tumor bearing mice, most of the IONPs was distributed in the liver and spleen, with obvious uptake in the tumor, less but significant amount in the kidney and brain. Taken together, multi-modal visualization approaches in our study provide detailed information on the cellular uptake and tissue distribution of IONPs from multiple levels and perspectives.