Thymosin beta-4 (Tβ4) is known to be ubiquitously involved in the actin monomer sequestering on the cytoskeleton. Our previous study showed specific temporal and special in situ expression pattern of Tβ4 mRNA in dental epithelial and mesenchymal cells in the developing tooth germ of the mouse lower first molar. In this study, we examined the functional implications of Tβ4 in the developmental course of the mouse lower first molar. An inhibition assay using Tβ4 antisense sulfur-substituted oligodeoxynucleotide (AS S-ODN) in cultured embryonic day 11.0 (E11.0) mandibles showed a significant growth inhibition of the tooth germ. However, no growth arrest of the cultured E15.0 tooth germ was observed by using Tβ4 AS S-ODN. The Tβ4 knockdown led to significantly decreased expression levels of type II/III runt-related transcription factor 2 (Runx2) and nucleolin (Ncl) in the cultured E11.0 mandibles. Since our previous studies proved that the inhibition of type II/III Runx2 and Ncl translations resulted in the developmental arrest of the tooth germ in the cultured E11.0 mandible, Tβ4 appears to play roles in tooth germ development via the regulation of the type II/III Runx2 and Ncl expressions. Tβ4 knockdown also resulted in decreased secretion of matrix metalloproteinase (Mmp)-2, a reduced cell motility activity and upregulation of E-cadherin in dental epithelial mDE6 cells. These results suggest that Tβ4 plays multiple functional roles in odontogenic epithelial cells in the early stages of tooth germ development by regulating the expression of odontogenesis-related genes.