Lymphocyte enhancer-binding factor 1 (Lef1), a HMG-domain protein, is thought to play important roles in inductive tissue interaction during tooth development. Lef1 knockdown in mice causes arrest at the bud stage in tooth development. As this gene participates in the regulation of a large and diverse set of peptide growth factors in ectomesenchymal cell differentiation of dental papilla, Lef1 appears to be a key factor in odontoblast differentiation. However, the relationship between Lef1 and odontoblast differentiation is still unclear. To analyze the biological roles of Lef1 in regulating odontoblast differentiation, we transiently overexpressed or suppressed Lef1 in cultured dental pulp cells. Lef1-overexpressing cells expressed higher levels of dentin sialoprotein (DSPP), osteocalcin and alkaline phosphatase (ALP) mRNA and formed larger numbers of mineralized nodules compared to control cells. However, Msx-1 expression or cell proliferation was unaffected by overexpression of Lef1. To further examine the role of Lef1 in dental pulp cells, we knocked down Lef1 expression in dental pulp cells using short interfering RNA (siRNA). Transient expression of siRNA against Lef1 markedly reduced Lef1 mRNA levels, and Lef1-suppressed cells expressed lower levels of DSPP, osteocalcin and ALP mRNA compared to control cells. Furthermore, the formation of mineralized nodules was inhibited by siRNA against Lef1; however, neither Msx-1 expression or cell proliferation was inhibited by siRNA against Lef1. These results outline the role of Lef1 in accelerating odontoblast differentiation by regulating DSP and osteocalcin mRNA expression in dental pulp cells, confirming that Lef1 is a key factor for odontoblast differentiation.