Objective: The purpose of this study is to investigate the effects of Epimedium flavonoids (EF), which is extracted from a traditional Chinese Epimedium herb, and its effect on the proliferation and differentiation of neural stem cells (NSCs) in vitro. Methods: The single cells isolated from the hippocampi of 1 day old neonatal rats were cultured in a serum-free condition medium DMEM/F12 (1 : 1) with different concentrations of EF or 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml basic fibroblast growth factor (bFGF). After 7 and 28 days, the neurospheres' diameters were measured. The formed neurospheres were cultured in the differentiation medium containing EF or 10% fetal bovine serum (FBS). After 12 hours and 7 days, immunofluorescent studies for nestin, Musashi-1, BrdU, beta-III-tubulin, NF-200 and GFAP were performed. The number and lengths of 10-15 axons of NF-200 immunopositive cells were measured. Results: The results showed that the isolated cells had the ability to propagate as neurospheres in the medium with 200 and 400 mu g/ml EF, but without any EGF or bFGF, and the volume of neurospheres increase gradually from 7 to 28 days. In comparison with FBS control, the number of NF-200 positive neurons had significantly increased in the EF groups where the newborn neurons were morphologically more mature and able to migrate farther away from neurospheres than in the FBS control. Discussion: The results demonstrate that EF effectively promotes the proliferation and differentiation of NSCs in vitro, suggesting that EF may have new properties of regulating central nervous system function by neurogenesis.