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Serotonin (5-HT) and the serotonergic system have recently been indicated as modulators of adult hippocampal neurogenesis. In this study, we evaluated the role of 5-HT on the functional features in neurospheres derived from adult neural stem cells (ANSC). We cultured neurospheres derived from mouse hippocampus in serum-free medium containing epidermal (EGF) and type-2 fibroblast growth factor (FGF2). Under these conditions ANSC expressed both isoforms of tryptophane-hydroxylase (TPH) and produced 5-HT. Blocking TPH function by para-chlorophenylalanine (PCPA) reduced ANSC proliferation, which was rescued by exogenous 5-HT. 5-HT action on ANSC was mediated predominantly by the serotonin receptor subtype 5-HT1A and, to a lesser extent, through the 5-HT2C (receptor) subtype, as shown by selectively antagonizing these receptors. Finally, we documented a 5-HT-induced increase of ANSC migration activity. In summary, we demonstrated a powerful serotonergic impact on ANSC functional features, which was mainly mediated by 5-HT1A receptors.