Thymosin beta-4 (Tβ(4)) is a major actin-sequestering molecule that contributes to cell growth, differentiation, motility, survival, mitosis and angiogenesis. It is overexpressed in certain type of carcinoma and fibrosarcoma cell lines and is associated with metastatic potential. The aim of this study was to investigate the relationship between Tβ(4) expression and clinicopathologic features and VEGF status in gastrointestinal stromal tumors (GISTs). Retrospectively, 60 GISTs were re-examined and immunohistochemistry for Tβ(4) and VEGF was performed. Increased expression of Tβ(4) and VEGF was observed in 26 (43.3%) and in 19 (31.6%) of the tumors, respectively. Tβ(4) expression was positively correlated with VEGF expression (p < 0.01). Tβ(4) and VEGF expression were significantly associated with tumor size (p = 0.00 and p = 0.02, respectively) and high mitosis (p = 0.03 and p = 0.00, respectively). Although Tβ(4) expression was positively associated with pleomorphism (p = 0.01), VEGF expression was positively associated with necrosis (p = 0.03). Tβ(4) expression was related with local recurrence and/or metastasis (p = 0.03), but VEGF expression was not (p = 0.12). We firstly demonstrate the presence of Tβ(4) protein in GISTs. Our study reveals that increased expression of Tβ(4) could be considered as an indicator of aggressive behavior of tumor.