Thymosin β4 (Tβ4) is expressed in developing tissue, where it stimulates cell differentiation and migration. Further, Tβ4 is expressed during molar development in mice, but the expression and function of Tβ4 in odontoblasts during mammalian tooth development have not yet been reported. Therefore, this study examined the expression and function of Tβ4 in differentiating odontoblasts during tooth development. As observed by immunohistochemistry, Tβ4 was expressed in the oral epithelium and inside cells of the tooth bud on embryonic day 15 (E15). Further, on E17, Tβ4 was expressed strongly in the dental lamina and oral epithelium, but only expressed in part of the cells in the outer and inner dental epithelium. Tβ4 was strongly expressed in the entire cytoplasm of odontoblasts on postnatal day 1 (PN1) and expressed intensively in the apical area of odontoblasts on PN4. Further, expression of Tβ4 was increased gradually in odontoblasts from PN1 to PN21. In an odontoblast cell line, MDPC-23, expression of Tβ4 mRNA and protein was increased strongly on day 4 and gradually decreased from day 14. The gene expression of dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP), osteocalcin (OCN), osteonectin (ON), and collagen type I, related with mineralization, was significantly decreased in si-Tβ4/MDPC-23 during differentiation compared to that in MDPC-23 cells. Taken together, our results suggest that Tβ4 may be involved in oral epithelial cell proliferation at the initial stage of tooth development and regulates the expression and secretion of proteins during odontoblast differentiation.