Objective To investigate the effects of hypoxic conditioned medium (HCM) of cerebral cortex cells on the proliferation of neural stem cells (NSCs) and to discuss the role of phosphoinositide 3-kinase (PI3-K) and c-Jun N-terminal kinase (JNK) signal pathways in the process. Methods The cerebral cortex cells of newborn SD rats (within 24 h after birth) were primarily cultured for 5 days, the cells were then cultured under 4% O₂, 1% O₂ or normal oxygen for 6 h, and the cultured media were collected as the HCM and normoxic conditioned medium (NCM). The specific inhibitors of PI3-K and JNK (LY294002 and SP600125) were added into the conditioned medium to culture NSCs. And then immunofluorescence staining was performed to identify NSCs in the neurospheres. The proliferation efficiency(number of neurospheres) and speed (diameter of neurospheres) were used to analyze the effects of hypoxic conditioned media on the proliferation of NSCs. Results The proliferation efficiencies of 4% HCM and NCM groups were significantly higher than that of 1% HCM group(P<0.01), and there was no significant difference between former two groups. The proliferation speed of two HCM groups was significantly faster than that of the NCM group(P<0.01), with the speed of 4% HCM group being the fastest one. The two inhibitors inhibited both the proliferation efficiency and speed of NSCs in the 4% HCM group(P<0.01), and the inhibitory effect of LY294002 was more prominent after 24 h. Conclusion 4% HCM of cerebral cortex cells can promote the proliferation speed of NSCs, and PI3-K pathway may play an important role in it.