Thymosin beta-10 (TB10) is a small G-actin binding protein that induces depolymerization of intracellular F-actin pools by sequestering actin monomers. Previously, we demonstrated that overexpression of TB10 in ovarian tumor cells increased the rate of cell death. As an initial step to define molecular mechanism of TB10-dependent apoptotic process in ovarian tumor cells, we searched a human ovary cDNA library for a novel TB10 binding protein using a yeast two-hybrid system. The selected protein was human E-tropomodulin (E-Tmod), another component of the actin binding proteins. Subsequently, two interacting protein components were determined quantitatively. Results showed that the full-length TB10 is required to bind with E-Tmod, and the TB10 binding site on E-Tmod partially overlaps with the actin binding site on E-Tmod. Moreover, introduction of E-Tmod cDNA into a tumor cell line reversed TB10 mediated apoptosis and restored actin architectures. These results may suggest that TB10 regulates apoptotic homeostasis by not only just binding to actin but also competing or blocking the protein complex formation of E-Tmod with actin.