The promotion of neurogenesis is considered to be an effective therapeutic strategy for neuropsychiatric disorders because impairment of neurogenesis is associated with the onset and progression of these disorders. We have previously demonstrated that orally ingested ergothioneine (ERGO), a naturally occurring antioxidant and hydrophilic amino acid, promotes neurogenesis in the hippocampal dentate gyrus (DG) with its abundant neural stem cells (NSCs) and exerts antidepressant-like effects in mice. Independent of its antioxidant activities, ERGO induces in cultured NSCs this differentiation through induction of the basic helix-loop-helix transcription factor Math1. However, the upstream signaling of Math1 in the mechanisms underlying ERGO-induced neuronal differentiation remains unclear. The purpose of the present study was to elucidate the upstream signaling with the aim of discovering novel targets for the treatment of neuropsychiatric disorders. We focused on neurotrophic factor signaling, as it is important for the promotion of neurogenesis and the induction of antidepressant effects. We also focused on the signaling of the mammalian target of rapamycin (mTOR) complex 1 (mTORC1), a known amino acid sensor, and the members of this signaling pathway, mTOR and p70 ribosomal protein S6 kinase 1 (S6K1). Exposure of cultured NSCs to ERGO significantly increased the expression of phosphorylated S6K1 (p-S6K1) at Thr389 in only 1 h, of phosphorylated mTOR (p-mTOR) in 6 h, and of the gene product of neurotrophin 4/5 (NT5) which activates tropomyosin receptor kinase B (TrkB) in 24 h. ERGO increased the population of βIII-tubulin-positive neurons, and this effect was suppressed by the inhibitors of S6K1 (PF4708671), mTORC1 (rapamycin), and TrkB (GNF5837). Oral administration of ERGO to mice significantly increased in the DG the expression of p-S6K1 at Thr389, the gene product of NT5, and phosphorylated TrkB but not that of p-mTOR. Thus, neuronal differentiation of NSCs induced by ERGO is mediated, at least in part, through phosphorylation of S6K1 at Thr389 and subsequent activation of TrkB signaling through the induction of NT5. Thus, S6K1 and NT5 might be promising target molecules for the treatment of neuropsychiatric disorders. Copyright © 2018. Published by Elsevier Inc.