Thymosin-beta-4 (TB4) as an actin-sequestering peptide has been detected outside of cells in blood plasma or in wound fluid. TB4 induces tumor metastasis and paclitaxel resistance, which is the most significant obstacle to successful therapy in tumors. Here we investigated the inhibitory effect of TB4 peptides on tumor cell death by paclitaxel. The effect of TB4 peptides was assayed by the measurement of caspase-3 activity, G2/M arrest, and Bcl-2 phosphorylation. Cell survival rate was increased and caspase-3 activity was decreased by the treatment with TB4 peptides. In contrast, small interfering RNA (siRNA) of TB4 inhibited cell viability and augmented caspase-3 activity. Significant changes in Bcl-2 phosphorylation were detected by TB4 peptide treatment or by the overexpression of TB4 gene in Hela cells. The reduced population in G2/M phase by TB4 peptide treatment was correlated with the decreased expression of cyclin B1. The data were confirmed in gastric tumor cell lines, SNU 638 (low TB4 level) and SNU 668 (high TB4 level), which were established from clinically isolated gastric tumors. In conclusion, soluble TB4 peptides produced in cancer cells could be an obstacle to treat tumors with paclitaxel. Therefore, TB4 could be a novel target to control paclitaxel resistance.