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Amelogenin (AMG) is a highly conserved protein secreted by ameloblasts. Some research indicates that AMG might induce the differentiation and maturation of odontoblasts. The aim of this study was to clarify the function of AMG during the differentiation of odontoblast-like MDPC-23 cells. The results revealed that the alkaline phosphatase activity and the number of mineralized nodules were significantly enhanced in AMG-overexpressing MDPC-23 cells during the mineralization process. Tissue-specific markers such as dentin matrix protein 1 and dentin sialophosphoprotein also elevated significantly, indicating the cell differentiation and maturation process. Furthermore, AMG could upregulate the phosphorylation levels of ERK1/2 and p38 MAPK. However, JNK, another MAPK pathway molecule, didn't change the activity at all. And the differentiation induced by AMG was abrogated when the MDPC-23 cells were treated with U0126 and SB203580, the inhibitors of ERK1/2 and p38, respectively. Taken together, our present results showed that AMG could promote the differentiation of odontoblast-like MDPC-23 cells via ERK1/2 and p38 MAPK pathways.