INTRODUCTION: Nel-like molecule-1 (Nell-1) with epidermal cell growth factor-like coding structures is a recently discovered secreted protein that can specifically induce osteoblast differentiation, bone formation, and bone regeneration. The expression and localization of Nell-1 during murine molar development suggest that this protein participates in odontoblastic differentiation. This study aims to assess the expression of Nell-1 in the pulp-dentin complex of human teeth and determine the effect of human recombinant Nell-1 (hrNell-1) on odontoblastic differentiation of cultured human dental pulp cells. METHODS: Immunohistochemical assay was performed to detect the expression and location of Nell-1 in healthy human teeth. The activity of alkaline phosphatase in human dental pulp cells stimulated by hrNell-1 was examined. Moreover, the expression of related odontoblastic markers was evaluated using quantitative real-time polymerase chain reaction and Western blot. RESULTS: Immunohistochemical results showed that Nell-1 was primarily expressed in the odontoblasts (odontoblastic bodies and processes), pulp fibroblasts, and endothelial cells of the blood vessels in human teeth. In vitro experiments showed that hrNell-1 could increase the activity of alkaline phosphatase and enhance the expression of important odontoblastic markers including osteopontin and dentin matrix protein 1 in cultured human dental pulp cells. CONCLUSIONS: Nell-1 plays a role in odontoblastic differentiation of human dental pulp cells and dentin formation.