► l-Cysteine stimulated proliferation and differentiation of NSCs. ► l-Cysteine-induced proliferation was associated with phosphorylation of ERK1/2. ► l-Cysteine-induced differentiation was due to the differentiation-related genes. ► AOAA or CBS siRNA attenuated the effects of l-cysteine on proliferation and differentiation of NSCs. Growing evidence has suggested that hydrogen sulfide (H2S) acts as a novel neuro-modulator and neuroprotective agent; however, it remains to be investigated whether H2S has a direct effect on neural stem cells (NSCs). We report here that NSCs expressed cystathionine β synthase (CBS) and addition of exogenous H2S donor, l-cysteine, stimulated proliferation and increased the differentiation potential of NSCs to neurons and astroglia. Moreover, pre-treatment with aminooxyacetic acid, the inhibitor of CBS or knockdown of CBS in using siRNA, significantly attenuated the effects of l-cysteine on elevated H2S levels and the cell proliferation; it also effectively suppressed l-cysteine-induced neurogenesis and astrocytogenesis. Further analysis revealed that l-cysteine-induced proliferation was associated with phosphorylation of extracellular signal-regulated kinases 1/2 and differentiation with altered expression of differentiation-related genes. Taken together, the present data suggest that l-cysteine can enhance proliferation and differentiation of NSCs via the CBS/H2S pathway, which may serve as a useful inference for elucidating its role in regulating the fate of NSCs in physiological and pathological settings.