Glutathione-modified superparamagnetic iron oxide nanoparticles (GSH-SPIONs) were prepared by conjugating glutathione (GSH) on the surface of the PEG (Polyethylene glycol)/PEI (polyethyleneimine)-SPIONs which were synthesized by thermal decomposition method. Thermogravimetric analysis showed that the mass fraction of GSH on the surface of SPIONs was 30.64 wt%. GSH-SPIONs in PBS were injected into the substantia nigra of rat brains. The subcellular distributions of the nanoparticles in the brains was examined by the transmission electron microscope (TEM). A remarkable amount of GSH-SPIONs were found in vesicles inside cell bodies and axons, and in mitochondria. TEM pictures show that GSH-SPIONs enter the neuronal cells by endocytosis and travel through axoplasmic transport. GSH-SPIONs have great potential as drug delivery agents in the brain to treat diseases or study brain function via mitochondria-targeting way or axoplasmic transport way.