The stem cells of human exfoliated deciduous teeth (SHEDs) are considered to be one of the main sources of seed cells in stem cell therapy. The aim of this study was to examine the effect of ciliary neurotrophic factor (CNTF) on neurogenic differentiation of SHEDs. With the consent of parents, SHEDs from 6 to 8 year old children were isolated and cultured. The mesenchymal stemness and the potential of multidirectional (adipogenic and osteogenic) differentiation for the isolated SHEDs were firstly determined. The effect of CNTF on specific neurogenic differentiation of SHEDs was then examined by detecting the expression of marker genes and proteins via RT-PCR, immunoblotting, and immunofluorescence microscopy. The isolated SHEDs expressed specific surface markers of mesenchymal stem cells, and their potential of osteogenic and adipogenic differentiation were confirmed. CNTF promoted the differentiation of SHEDs into neuron-like cells with a high expression of acetylcholine transferase (CHAT), a marker of cholinergic neurons. The expression of other neuron markers including nestin, microtubule-associated protein 2 (MAP 2), and β-tublin III was also detected. Interestingly, the expression of neurogenic markers was maintained at a high level after neurogenic induction. SHEDs can be induced by CNTF to differentiate into cholinergic neuron-like cells under appropriate culture conditions. Our findings have laid a foundation for future use of SHEDs to treat neurological diseases. © 2020, The Author(s).