Thymosin β10 is a cytoplasm G-actin sequestering protein whose functions are largely unknown. To determine the direct effects of exogenous thymosin β10 on angiogenic potentials as endothelial cell migration and capillary-like tube formation, human coronary artery endothelial cells (HCAECs) were incubated with increasing doses of thymosin β10 (25–100 ng/ml). By using a modified Boyden chamber assay, thymosin β10 inhibited cell migration in a dose- and time-dependent manner with the maximal effect being a 36% reduction at 100 ng/ml as compared to controls (P < 0.01). In addition, thymosin β10 (100 ng/ml) significantly inhibited the capillary-like tube-formation of HCAECs on Matrigel, showing a 21% reduction of the total tube length as compared to negative controls (P < 0.01). Furthermore, by using real time PCR analysis, thymosin β10 significantly decreased mRNA levels of vascular endothelial growth factor (VEGF), VEGF receptor-1 (VEGFR-1) and integrin αV after 24 h treatment in HCAECs. By contrast, thymosin β4 significantly increased HCAEC migration. These results indicate that thymosin β10, but not thymosin β4, have direct inhibitive effects on endothelial migration and tube formation that might be mediated via downregulation of VEGF, VEGFR-1 and integrin αV in HCAECs. This study suggests a potential therapeutic application of thymosin β10 to the diseases with excessive angiogenesis such as cancer. Cell Motil. Cytoskeleton 63: 2006. © 2006 Wiley-Liss, Inc.