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The toxicity of superparamagnetic iron oxide nanoparticles (SPIONs) is still a vital topic of debate and the mechanisms remain unclear. In the present study, overdose SPIONs could induce osteosarcoma cell death and the effects were exaggerated when combined with spinning magnetic field (SMF). In the combination group, mitochondrial transmembrane potential decrease more obviously and reactive oxygen species (ROS) was found to generate much higher in line with that of the apoptosis ratio. Meantime, amount of autophagy was induced. Inhibiting the autophagy generation by 3-methyladenine (3-MA) increase cell viability but decrease the caspase 3/7 and caspase 8 activities in combination groups, and inhibiting apoptosis took the same effect. In the end, the SPIONs effects on xenograft mice was examed by intratumoral injection. The result showed that the combination group could greatly decrease the tumor volume and prolong the lifespan of mice. In sum, the result indicated that overdose SPIONs induced ROS generation, and excessive ROS induced by combination of SPIONs and SMF contribute to autophagy formation, which play a apoptosis-promoting role that formed as a platform to recruits initiate the caspase activities.