Odontoblasts, which derive from dental papilla, are a type of terminally differentiated matrix-secreting cells. Previous studies have identified various transcription factors involved in the differentiation process of odontoblasts. We have recently found that Krüppel-like factor 4 (Klf4) was expressed in the polarizing and elongating odontoblasts, but the function of Klf4 in the differentiation of odontoblasts is still unclear. We hypothesized Klf4 promoted the differentiation of odontoblasts by up-regulating some odontoblast-related genes. In this study, we found that the expression of Klf4 increased significantly during the odontoblastic differentiation of primary mouse dental papilla cells and the mouse dental papilla cell line-mDPC6T. Overexpression of Klf4 significantly up-regulated odontoblast-related genes, such as Dmp1, Dspp, and Alp, and promoted the accumulation of mineral nodules. Knock-down of Klf4 down-regulated expression of Dmp1, Dspp, and Alp, and inhibited mineral deposition. We applied in silico analysis and identified one target gene of Klf4Dmp1. Based on further analysis of ChIP data, EMSA and dual luciferase activity assays, we confirmed that Klf4 was able to specifically bind to the Dmp1 promoter and transactivate its expression. Furthermore, forced expression of Dmp1 in the Klf4 knock-down mDPC6T cell line significantly recovered its odontoblastic differentiation ability. Our data confirmed our hypothesis that Klf4 promotes the differentiation of odontoblasts via the up-regulation of Dmp1. J. Cell. Physiol. 228: 2076–2085, 2013. © 2013 Wiley Periodicals, Inc.