Bioassay-guided fractionation, combined with screening based on EGF-responsive neural stem cells (NSCs) differentiation assay, has been used to search for active molecules from Panax notoginseng. Ginsenosides Rg3 (1), Rk1 (2), and Rg5 (3) were identified as potential neurogenic molecules. The degrees of their neurogenic effects were found to be 3 > 2 > 1. The neurogenic effect of 3 represents a biphasic dose- and time-dependent regulation. Transient exposure of NSCs to 8 microM 3 for 24 h followed by 1 microM and 72 h incubation was the optimal procedure for the induction of neurons in NSCs, and compound 3 resulted in an approximately 3-fold increase in neurogenesis at the expense of astrogliogenesis. The neurogenic effect of 3 was completely eliminated by the Ca2+ channel antagonist nifedipine. These findings imply that 3 may be utilized as a pharmacological agent in studying the molecular regulation of neurogenesis of brain stem cells and, subsequently, for treatment of neurodegenerative diseases.