S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the synthesis of polyamines essential for cell growth and proliferation. Its overexpression induces the transformation of murine fibroblasts in both sense and antisense orientations, yielding highly invasive tumors in nude mice. These cell lines hence provide a good model to study cell invasion. Here, the gene expression profiles of these cells were compared with their normal counterpart by microarray analyses (Incyte Genomics, Palo Alto, CA, and Affymetrix, Santa Clara, CA). Up-regulation of the actin sequestering molecule thymosin β4 was the most prominent change in both cell lines. Tetracycline-inducible expression of thymosin β4 antisense RNA caused a partial reversal of the transformed phenotype. Further, reversal of transformation by dominant-negative mutant of c-Jun (TAM67) caused reduction in thymosin β4 mRNA. Interestingly, a sponge toxin, latrunculin A, which inhibits the binding of thymosin β4 to actin, was found to profoundly affect the morphology and proliferation of the AdoMetDC transformants and to block their invasion in three-dimensional Matrigel. Thus, thymosin β4 is a determinant of AdoMetDC-induced transformed phenotype and invasiveness. Up-regulation of thymosin β4 was also found in ras-transformed fibroblasts and metastatic human melanoma cells. These data encourage testing latrunculin A-like and other agents interfering with thymosin β4 for treatment of thymosin β4-overexpressing tumors with high invasive and metastatic potential. ©2006 American Association for Cancer Research.