Background: Expression of the small peptide thymosin β4 is associated with angiogenesis induction, accelerated wound healing, and the metastatic potential of tumor cells. However, little is known about the mechanism(s) by which thymosin β4 promotes metastasis. Methods: Northern blot analysis and immunohistochemistry were used to examine thymosin β4 expression in mouse melanoma B16 cell lines and in B16-F10 cells derived from metastatic mouse lung tumors, respectively. B16-F10 cells infected with adenoviruses containing a thymosin β4 expression vector or an empty vector were injected subcutaneously and intravenously into C57BL/6 mice to evaluate tumor growth and metastatic potential, respectively. In vitro assays were used to study cell migration, invasion, matrix metalloproteinase activity, cell proliferation, and angiogenic activity of adenovirus-infected B16-F10 cells. Statistical significance of all results was analyzed by two-tailed Student’s t tests. Results: Thymosin β4 mRNA was expressed in primary cultured B16-F10 cells derived from lung metastases and in B16-F10 cells that had formed lung tumors after being injected into mice but not in the B16-F1, B16-F10, or B16-BL6 cell lines. The mean tumor sizes in mice 20 days after injection with B16-F10 cells infected with thymosin β4–expressing adenovirus and with control adenovirus were 21.7 mm (95% confidence interval [CI] = 17.7 to 25.7 mm) and 13.3 mm (95% CI = 11.1 to 15.3 mm), respectively (difference = 8.4 mm; P = .036). The mean numbers of metastatic lung nodules in mice (n = 20) 2 weeks after intravenous injection with thymosin β4–expressing adenovirus and with control adenovirus were 46.7 (95% CI = 35.0 to 57.7) and 10.9 (95% CI = 6.2 to 15.6), respectively (difference = 35.8 metastatic lung nodules, P<.001). Thymosin β4 overexpression was associated with a mean 2.3-fold increase (95% CI = 1.9- to 2.7-fold increase; P<.001) in B16-F10 cell migration and a mean 4.4-fold increase (95% CI = 3.3- to 5.5-fold increase; P<.001) in the number of blood vessels in solid tumors derived from injected B16-F10 cells but had no effect on cell invasion, proliferation, or matrix metalloproteinase activity. This induction of angiogenesis by thymosin β4 was associated with induction of vascular endothelial growth factor expression. Conclusion: Thymosin β4 may stimulate tumor metastasis by activating cell migration and angiogenesis.