Background: Disrupted iron homeostasis in the substantia nigra (SN) is involved in Parkinson's disease (PD). In our previous studies, we reported that in neurons, 6-hydroxydopamine (6-OHDA) activated iron regulatory protein 1 (IRP1), increased the expression of divalent metal transporter-1 (DMT1) and decreased the expression of ferroportin 1 (FPN1), leading to increased iron influx and decreased iron efflux. In astrocytes, 6-OHDA increased both DMT1 and FPN1 expression, increased iron traffic. However, the mechanisms remain elusive. Hypoxia-inducible factors (HIFs) are known to be important in regulating iron homeostasis. Objective: To explore that the mechanism of iron metabolism in astrocytes treated with 6-OHDA. Materials and Methods: Using primary cultured rat astrocytes and western blots. Results: We observed that protein levels of HIF-1α and HIF-2α in astrocytes were significantly increased by treatment with 6-OHDA, whereas expression did not change in ventral mesencephalic (VM) neurons. Furthermore using inhibitors of HIF-1α and HIF-2α, we observed that HIF-2α inhibitor markedly reduced the up-regulation of DMT1 and FPN1 by 6-OHDA and decreased ferrous iron influx and efflux. However, there was no effect following treatment of astrocytes with HIF-1α inhibitor. Activation of HIF-2α by Phorbol 12-myristate 13-acetate (PMA) and 6-OHDA was blocked by bisindolylmaleimide I hydrochloride (Bisl) and N-acetyl-l-cysteine (NAC) or Nω-Nitro-L-arginine methyl ester hydrochloride (L-NAME) preventing downstream activation of DMT1 and FPN1. NAC and L-NAME both block the the activation of PKCδ phosphorylation by 6-OHDA in primary cultured astrocytes. Conclusion: Our data indicate that HIF-2α, but not HIF-1α, regulates expression of DMT1 and FPN1 in astrocytes, and PKC pathway is involved in 6-OHDA activation of HIF-2α in astrocytes, which may be associated with ROS and NO activation of PKCδ.