Icariin (ICA), which is an essential bioactive component extracted from the herb Epimedium, possesses neuroprotective properties. The aim of the present study was to investigate the regulatory roles of ICA in cell proliferation and gene expression in human neural stem cells (NSCs) in vitro. Single cells were isolated from the corpus striatum of 16-20-week human fetuses obtained following spontaneous abortion. The cells were cultured in Dulbecco's modified Eagle's medium/F12 complete medium and were characterized by immunostaining and cell differentiation assay. NSCs were treated with ICA, and cell proliferation was assessed using the Cell Counting kit-8 cell proliferation assay kit. In addition, neurosphere formation was comparatively studied between the ICA-treated and control cells. cDNA microarray analysis was performed to examine the effects of ICA on gene expression. Altered expression of genes important for regulating NSC proliferation was further analyzed by quantitative polymerase chain reaction (qPCR). The results demonstrated that typical neurospheres appeared after 7-10 days of culturing of individual cells isolated from the corpus striatum. These cells expressed nestin, an important NSC marker, and in the presence of differentiation medium they expressed beta-III-tubulin, a specific neuronal marker, and glial fibrillary acidic protein, an astrocyte marker. Treatment with ICA enhanced NSC proliferation and the formation of neurospheres. Microarray data and pathway analysis revealed that the genes regulated by ICA were involved in several signaling pathways, including the Wnt and basic fibroblast growth factor (bFGF) pathways, which are important for the regulation of NSC function. Upregulation of frizzled class receptor 7 and dishevelled segment polarity protein 3, which are key players in the Wnt pathway, and fibroblast growth factor receptor 1, which is the receptor for bFGF, and downregulation of glycogen synthase kinase-3 beta, which is a Wnt pathway inhibitor, was further validated by qPCR. In conclusion, ICA promoted proliferation and regulated gene expression in human NSCs, thus suggesting that ICA may exert its neuroprotective effects by regulating NSC activity.