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We recently reported that intermittent hypoxia facilitated the proliferation of neural stem/progenitor cells (NPCs) in the subventricule zone and hippocampus in vivo. Here, we demonstrate that hypoxia promoted the proliferation of NPCs in vitro and that hypoxia-inducible factor (HIF)-1α, which is one of the key molecules in the response to hypoxia, was critical in this process. NPCs were isolated from the rat embryonic mesencephalon (E13.5), and exposed to different oxygen concentrations (20% O2, 10% O2, and 3% O2) for 3 days. The results showed that hypoxia, especially 10% O2, promoted the proliferation of NPCs as assayed by bromodeoxyuridine incorporation, neurosphere formation, and proliferation index. The level of HIF-1α mRNA and protein expression detected by RT-PCR and western blot significantly increased in NPCs subjected to 10% O2. To further elucidate the potential role of HIF-1α in the proliferation of NPCs induced by hypoxia, an adenovirus construct was used to overexpress HIF-1α, and the pSilencer 1.0-U6 plasmid as RNA interference vector targeting HIF-1α mRNA was used to knock down HIF-1α. We found that overexpression of HIF-1α caused the same proliferative effect on NPCs under 20% O2 as under 10% O2. In contrast, knockdown of HIF-1α inhibited NPC proliferation induced by 10% O2. These results demonstrated that moderate hypoxia was more beneficial to NPC proliferation and that HIF-1α was critical in this process.