D-Serine, the endogenous co-agonist of N-methyl-D-aspartate (NMDA) receptors, has been recognized as an important gliotransmitter in the mammalian brain. D-serine has been shown to prevent psychostimulant-induced decrease in hippocampal neurogenesis. However, the mechanism whereby D-serine regulates neurogenesis has not been fully characterized. Therefore, this study was designed to investigate the impacts of D-serine on the proliferation, migration, and differentiation of primary cultured neural stem cells (NSCs). Immunohistochemistry analysis revealed NSCs expressed D-serine as well as serine racemase (SR). Degradation of endogenous D-serine with D-amino acid oxidase (DAAO) significantly inhibited the proliferation and neuronal differentiation of NSCs, but failed to affect their radial migration. Reversely, addition of exogenous D-serine did not affect the proliferation and migration of NSCs, but promoted NSC differentiation into neurons. Furthermore, DAAO could suppress the amplitude of glutamate-induced Ca(2+) transient, and thereby, inhibited the phosphorylation of glycogen synthase kinase3β (GSK3β), extracellular signal-regulated kinases1/2 (ERK1/2), and cAMP-responsive element-binding protein (CREB). Our findings demonstrate for the first time that NSCs can synthesize D-serine and, thereby, promote themselves proliferation and neuronal differentiation, which may afford a novel therapeutic strategy for the neurological disorders that require nerve cell replenishment, such as neurodegenerative diseases and stroke.