The temperature variation rule of nano-magnetic fluid in the specific magnetic field and the effect on the treatment of malignant glioma were examined. The temperature variation of nano-magnetic fluid in the specific magnetic field was investigated by heating , and cell morphology was observed through optical microscopy and electron microscopy. MTT detection also was used to detect the effect of FeO nanometer magnetic fluid hyperthermia (MFH) on the proliferation of human U251 glioma cell line. The FeO nano MFH experiment was used to detect the inhibition rate of the tumor volume in nude mice with tumors. The results of the experiment showed that the heating ability of magnetic fluid was positively correlated with its concentration at the same intensity of the magnetic field. The results also indicated the prominent inhibitory effect of nanometer MFH on the proliferation of glioma cells, which was a dose-dependent relationship with nanometer magnetic fluid concentration. The hyperthermia experiment of nude mice with tumors displayed a significant inhibiting effect of FeO nanometer magnetic fluid in glioma volume. These results explain that iron (II, III) oxide (FeO) nanometer MFH can inhibit the proliferation of U251 glioma cells, and has an obvious inhibitory effect on glioma volume, which plays a certain role in the treatment of brain glioma.