Background: Tanshinone IIA is a lipid-soluble compound extracted from Chinese herb Danshen which was commonly used in the treatment of cerebrovascular diseases. Brain iron homeostasis is very essential for normal physiological functions of neurons, and brain iron accumulation contributes to many neurological disorders. The present study was aimed to determine whether Tanshinone IIA protects against cerebral ischemic injury via maintaining brain iron homeostasis. Methods: Wistar Rats were orally administered with Tanshinone IIA (4, 20 and 100 mg/kg/d) for one week, and then subjected to cerebral ischemia by middle cerebral artery occlusion (MCAO) for 2 h. In vitro, cultured neurons were pretreated with Tanshinone IIA (2, 10 and 50 uM) for one week, and then exposed to hypoxia for 24 h. Brain iron content was determined by Flame Atomic Absorption Spectrophotometer and intracellular free iron level was measured by laser scanning Confocal microscopy. Expression levels of iron transport proteins including DMT1, TfR, Fpn1 and Heph were assessed by Western blot technique. Results: Tanshinone IIA pretreatment resulted in a significant reduction of cerebral infarct volume in MCAO rats. Compared with control rats, cerebral ischemia considerably augmented total iron content in the brain of MCAO rats, which was also effectively restricted by Tanshinone IIA pretreatment. MCAO rats exhibited the increased expression of iron influx proteins DMT1 and TfR, and the decreased expression of iron efflux proteins Fpn1 and Heph compared with control rats, which was responsible for elevated iron content in the ischemic brain. Tanshinone IIA pretreatment prevented the dysregulation of these four iron transport proteins and maintained brain iron homeostasis. In vitro studies also confirmed that Tanshinone IIA alleviated the hypoxia-induced decline of cell viability and the overload of intracellular free iron level in neurons through downregulating the expression of DMT1 and TfR, and upregulating the expression of Fpn1 and Heph. Conclusion: Tanshinone IIA protected brain tissues against ischemic and hypoxic damage in vivo and in vitro by mediating brain iron homeostasis which was associated with the downregulation of DMT1 and TfR, and the upregulation of Fpn1 and Heph. These results provide new insights into molecular mechanisms of ischemia-induced brain iron abnormalities and suggest maintaining brain iron homeostasis may be a novel therapeutic strategy for ischemic cerebrovascular diseases.