Glutamate is an excitatory transmitter and can induce neurotoxicity, it can also increase the iron concentrations in the brain, but little is known about the detailed molecular regulation mechanism of iron metabolism by Glu. Based on our previous data, iron metabolism related proteins might be associated with the increase of brain iron contents induced by neurotransmitter. To investigate the issues, the iron contents, non-transferrin-bound iron (NTBI) uptake and the expression of iron uptake and iron release proteins were firstly examined in vivo and in vitro with iron histochemistry, inductively coupled plasma mass spectroscopy (ICP-MS), 55 Fe radioactive liquid scintillation counting and western blot methods. Data showed that glutamate induced the increase of total iron contents, storage iron contents and NTBI uptake activity. Moreover, only divalent metal transporter 1, one of iron uptake proteins, was increased in rat brain and PC12 cells treated with glutamate. Further investigations revealed that nuclear factor кB (NF-кB) and protein kinase C (PKC) were involved in the regulation of DMT1 in PC12 cells treated with glutamate. These findings demonstrate that glutamate increases iron contents in the brain through increased NTBI, and that DMT1 is the key molecule underlying regulation of iron metabolism by glutamate, Furthermore, NF-кB and PKC play important roles in the regulatory pathway of DMT1 expression by glutamate. Thus, it implicates that inhibiting the expression of DMT1 and disruption of its regulation pathway might be effective strategies in attenuating glutamate neurotoxicity through decreased iron contents.