Thymosin beta(10) is a monomeric actin sequestering protein that regulates actin dynamics. Previously, we and others have shown that thymosin beta(10) acts as an actin-mediated tumor suppressor. In this study, we show that thymosin beta(10) is not only a cytoskeletal regulator, but that it also acts as a potent inhibitor of angiogenesis and tumor growth by its interaction with Ras. We found that overexpressed thymosin beta(10) significantly inhibited vascular endothelial growth factor-induced endothelial cell proliferation, migration, invasion, and tube formation in vitro. Vessel sprouting was also inhibited ex vivo. We further show that thymosin beta(10) directly interacted with Ras. This interaction resulted in inhibition of the Ras downstream mitogen-activated protein kinase/extracellular signal-regulated kinase kinase signaling pathway, leading to decreased vascular endothelial growth factor production. Thymosin beta(10) injected into a xenograft model of human ovarian cancer in nude mice markedly inhibited tumor growth and reduced tumor vascularity. In contrast, a related thymosin family member, thymosin beta(4), did not bind to Ras and showed positive effects on angiogenesis. These findings show that the inhibition of Ras signal transduction by thymosin beta(10) results in antiangiogenic and antitumor effects, suggesting that thymosin beta(10) may be valuable in anticancer therapy.