Background: Phototherapy is an important method to treat vitiligo. However, it is unclear how phototherapy affects melanocyte precursors and skin neural crest stem cells. Objective: To investigate the underlying mechanisms of narrow- band ultraviolet B (NB-UVB) induced melanocyte lineage differentiated from human scalp-derived neural crest stem cells (HS-NCSCs). Methods: HS-NCSCs were expanded from scalp hair follicles. The c-Kit^-/CD57^- HS-NCSCs were isolated by cell sorting. Different doses of NB-UVB were used to irradiate these HS-NCSCs. Cell ultrastructure was examined by transmission electron microscope. Melanocyte marker expression was analyzed by Quantitative RT-PCR and Western blot. Cell proliferation and migration were also evaluated. Results: The c-Kit^-/CD57^- HS-NCSCs expressed embryonic NCSC biomarkers. NB-UVB at a dose of 100 mJ of NB-UVB had little effect on the cell proliferation of differentiated melanocytes from c-Kit^-/CD57^- HS-NCSCs, while 700 mJ inhibited cell proliferation significantly. The dendritic processes of differentiated melanocytes increased after radiation. The tyrosinase and Melanocortin 1 receptor (Mc1R) expression of differentiated melanocytes increased after NB-UVB exposure. The effect of NB-UVB on tyrosinase expression was modulated by signaling inhibitors H89 and PD98059 as well as Mc1R level in the cells. The migration ability of differentiated melanocytes was enhanced under 100 mJ exposure. Conclusion: These data demonstrate that NB-UVB facilitates melanocytic differentiation of the HSNCSCs and enhances migration of these cells. Mc1R and cAMP pathway play a critical role in NB-UVB induced melanocytic differentiation. (Ann Dermatol 32(4) 289∼297, 2020)