Because of its unique magnetic properties, the iron oxide (Fe 3 O 4 ) nanoparticle has been widely exploited and its application in various fields has promised immense benefits. However, doubts exist over the use of Fe 3 O 4 -nanoparticles in human beings. Thus, the aim of the current study was to find out the potential safety range of medical use. Twenty-five Kunming mice were exposed to Fe 3 O 4 -nanoparticles via intraperitoneal injection daily for 1 week at doses of 0, 5, 10, 20, and 40 mg/kg. Hepatic and renal tissues were sliced for physiological observation. Injuries were observed in the high-dose groups (20 and 40 mg/kg) compared with the control group (0 mg/kg). Biomarkers of reactive oxygen species, glutathione, malondialdehyde, DNAprotein crosslinks, and 8-hydroxy-2′-deoxyguanosine in the hepatic and renal tissues were detected. Injury to tissues and oxidative damage to cells at the molecular level was found. The safest dose recommended from the results of this study is 5 mg/kg, as we believe this to be an upper limit balancing the benefits and risks for sub-long-term exposure.