Ameloblastoma is regarded to be a benign odontogenic tumor, but it is destructive, locally invasive and presents a high rate of recurrence. Thymosin β4 (Tβ4) is closely associated with tooth germ development. Tβ4 also plays a role in malignant progression and invasion. However, little is known about the function of Tβ4 in odontogenic tumors. Thus, we investigated Tβ4 expression in ameloblastomas and compared it with odontomas. We immunohistochemically evaluated the expression of Tβ4, ameloblastin (AMBN), amelogenin (AMEL) and enamelin (ENAM) in 57 samples of ameloblastomas from 40 patients, and also assessed the expression of these molecules in 11 cases of odontomas, two of ameloblastic fibro-odontomas and one of tooth germ-like structures without the formation of enamel and dentin. Tβ4 signals were observed in almost all of the ameloblastomas. The signals were observed in both peripheral columnar cells and central polyhedral/angular cells. Similar findings were observed in tooth germ-like structures, and in the ameloblastomatous nests in the ameloblastic fibro-odontomas. These samples had negative results for AMBN, AMEL and ENAM. Meanwhile, Tβ4 signals were not seen in the odontomas, although immunolabeling for AMBN, AMEL and ENAM was observed in the enamel matrix and in some ameloblasts. Ectomesenhymal regions in the odontomas were negative for staining with the antibodies for AMBN, AMEL and ENAM. These results suggest that Tβ4 could be associated with morphogenesis and tumor invasion in the ameloblastoma, and that Tβ4 may play a role in the behavior of ameloblastoma.