Neural stem cells(NSCs) exist widely in the developing and adult mammalian brain, which are self-renewing and can differentiate into neurons, astrocytes or oligodendrocytes in vitro. The proliferation of NSCs in vitro has been reported to be regulated by various factors. However, growing studies by the use of oxygen microelectrodes show that the mammalian embryo developed in uterus with a hypoxic environment and neural stem or progenitor cell in the brain are also with hypoxic niche. The mean oxygen concentration is about 1-5% in tissues. To date, in vitro studies using NSCs have primarily been done under atmospheric conditions of 20% oxygen as the standard culture system. This indicates that traditional oxygen concentration in cell culture system may be a hyperoxia environment but not a condition of “physiological hypoxia” to most of cells in situ. Herein, we performed a series of experiments to testify whether the exogenous hypoxia could impact the growth of NSCs and their function? How hypoxia regulates the property of NSCs? And then what’s the molecular involved in this process? Firstly, we compared the different exogenous oxygen content on the NSCs’ growth in vitro. The embryo-derived neural stem cells were cultured under the 3%,10% and 20% oxygen concentration. There are about 2- to 5- fold increases in the number of NSCs cultured after exposure to hypoxia condition compared to normal condition. Especially, mild hypoxia dramatically promotes the proliferation of NSCs and decreases its apoptosis. The above results were further confirmed in both mouse and human-derived neural stem cells from embryo and adult, which revealed that neural stem cells in vitro prefer 1 to 10% oxygen culture environment. Thus, Mild hypoxia (1-10% oxygen) is a much more potent trigger to promote the proliferation of adult stem cells than normoxia (20% oxygen), suggesting mild hypoxia is a novel method for expansion of various adult stem cells in vitro. We further examine the differentiation ability of NSCs expanded under hypoxia conditions in vitro. The NSCs cultured in hypoxia (3% O2) displayed an increase in the percentage of neurons. Especially the percentage of TH-positive neurons differentiated from NSCs in lowered oxygen increased significantly. Dopamine (DA) content in supernatant of culture hypoxia group was the double of that in the normoxia group. This study may also offer a new approach to yield DA neurons by using a physical factor. The data above demonstrate that low oxygen significantly promotes the proliferation of NSCs and supports the property of self-renewal in vitro. However, the molecular mechanisms underlying this hypoxia-driven proliferation are unknown. To address this question, a cDNA microarray containing 5704 rat genes was used to characterize the gene expression pattern during hypoxia-driven proliferation of NSCs. Of the 5704 genes examined, 49 were down-regulated less than 0.5-fold and 22 were upregulated more than twofold at 24 h. At 72 h, 60 genes were upregulated and 11 were downregulated. Among the 71 differentially expressed genes identified at 24 h, the greatest number were involved in glycolysis and metabolism (36%), followed by transcriptional regulation (15%) and cell organization and biogenesis (10%). The NSCs under low oxygen consumed more glucose and produced energy by glycolysis. The information gained from gene expression and metabolic changes of NSCs under low oxygen conditions will provide new approaches for the evaluation of NSCs as potential in vivo cellular therapeutics. Without doubt, Hypoxia-inducible factor (HIF)-1, which is one of the key transcription factors under hypoxia, play an important role mediating a variety of adaptive cellular and systemic responses to hypoxia by regulating the expression of more than 50 different genes. The hypoxia-induced Small non-coding RNA (ncRNA) was investigated and shown involved in regulation of NSC proliferation. Results revealed that 15 small RNAs were up-regulated at least threefold and 11 were down-regulated in NSCs after subjected to hypoxic conditions. Especially, MiR-210 was observed to be highly expressed in NSCs in a time- and oxygen-dependent manner, and is directly regulated by HIF-1a. Hypoxia-induced expression of miR-210 may be involved in regulating apoptosis and proliferation of NSCs under hypoxia. In summary, our studies demonstrate that mild hypoxia not only promote the self-renew ability of NSCs in vitro, but also increase their differentiation ability into neurons. Therefore, via expansion of NSCs in vitro or modification of it’s’ property in situ, mild hypoxia could become a potential approach for allograft cell transplantation. We hope these findings will aid in the transplantation of NSCs to treat neural degenerative diseases such as Parkinson’s disease and brain trauma.