Docosahexaenoic acid (22:6n-3), one of the main structural lipids in the mammalian brain, plays crucial roles in the development and function of brain neurons. We examined the effect of docosahexaenoic acid on neuronal differentiation of neural stem cells in vitro and in vivo. Neural stem cells obtained from 15.5-day-old rat embryos were propagated as neurospheres and cultured under differential conditions with or without docosahexaenoic acid for 4 and 7 days. Docosahexaenoic acid significantly increased the number of Tuj1-positive neurons compared with the control on both culture days, and the newborn neurons in the docosahexaenoic acid group were morphologically more mature than in the control. Docosahexaenoic acid significantly decreased the incorporation ratio of 5-bromo-2'-deoxyuridine, the mitotic division marker, during the first 24 h period; it also significantly decreased the number of pyknotic cells on day 7. Thus, docosahexaenoic acid promotes the differentiation of neural stem cells into neurons by promoting cell cycle exit and suppressing cell death. Furthermore, dietary administration of docosahexaenoic acid significantly increased the number of 5-bromo-2'-deoxyuridine(+)/NeuN(+) newborn neurons in the granule cell layer of the dentate gyrus in adult rats. These results demonstrate that docosahexaenoic acid effectively promotes neurogenesis both in vitro and in vivo, suggesting that it has the new property of modulating hippocampal function regulated by neurogenesis.