Thymosin β4 (Tβ4) has been recently reported to play a role in dentinogenesis by regula-ting the expression of dentin matrix proteins. Based on previous studies, it is hypo-thesized that Tβ4 is associated with the formation of the enamel matrix and thus plays an important role in ameloblast. However, there is no report on the function of Tβ4 during tooth development so far. Therefore, in this study, we aimed to investigate the expression of Tβ4 and its function in ameloblasts during mouse tooth development. Tβ4 was expressed strongly in the tooth bud at the bud stage and in the dental lamina and oral epithelium at the cap stage. In advanced bell stage at postnatal day 4, large elongated ameloblasts were observed and the expression of the Tβ4 protein was the highest, with the enamel being was thicker than that in the early bell stage. The length of ameloblasts increased from the presecretory to the secretory stage and decreased from the maturation to the protective stage. These results suggest that Tβ4 participates not only in the proliferation of oral epithelial cells during the early stage of tooth development but also regulates enamel protein secretion in ameloblasts and enamel mineralization.