Objective：To prepare superparamagnetic iron oxide (SPIO) nanoparticles and to observe its acute toxicity on mice, so as to pave a way for further study on its longterm toxicity and on its role as a carrier in magnetic resonance gene imaging. Methods: The SPIO nanoparticle was obtained by means of coprecipitation, and its physical and chemical parameters were determined by transmission electron microscope, atomic force microscope, and 1.5 T super conduct MR, etc. According to the administration pathway and doses of SPIO, 90 mice were divided into oral administration (with a total dose of 2 104.8 mg/kg and a volume of 40 ml/kg, n=30), intravenous injection (a total dose of 438.5 mg/kg and a volume of 25 ml/kg, n=30) and intraperitoneal injection (with a total dose 1 578.6 mg/kg and a volume of 30 ml/kg, n=30) groups. Another 10 mice in each group receiving the same dose of normal saline via the same pathway served as the controls (n=10). The general condition, the major serologic parameters, and the pathological changes of major organs were observed 14 d after administration in each group. Results: We have successfully prepared SPIO, and its core component was Fe3O4 crystal, with a size of 2035 nm, a T2 relaxivity of 0.155×106 mol-1·sec-1, a specific saturated magnetization of 68.395 68 emu/g, and a retentivity of 21.463 74 Gs. There was no death of mice during the observation. There was no significant difference in serological parameters between mice of different groups and between each experiment group and their corresponding control group. No edema, degeneration and necrosis were seen in the liver, spleen, kidney, heart, and lungs by HE staining and marrow by Wright staining; only a few blue particles were observed in the liver and spleen in the administration groups by Prussian blue staining, none observed in the control groups. Conclusion: SPIO prepared in the present study meets the requirement of MR imaging, with no acute toxicity to mice, and warrants further study for future MR gene imaging.