Thymosin β4 has been reported to play the key roles in tumor growth, metastasis, and angiogenesis. Although the importance of thymosin β4 in angiogenesis and metastasis is known, few studies to show the expression patterns of thymosin β4 in human tissues including tumors have been conducted. The comparisons of the expression of thymosin β4 between the normal and tumor tissues are also needed to study the role of thymosin β4 in tumor formation. Using tissue microarray analysis, we compared the expression patterns of thymosin β4 in the normal human tissues and in the tumors to screen certain tumors and upregulated the expression of thymosin β4 by tumorigenesis. Thymosin β4 was highly expressed in the hepatic cells in the normal adult liver, duct, and acinar cells in pancreas, and muscle cells in the heart and also expressed highly in certain tumor cells, including osteosarcoma, colon adenocarcinoma, esophageal squamous cell carcinoma, kidney and urinary bladder transitional carcinoma, lung cancer, and liver cancer. Comparing the thymosin β4 expression between normal and tumors, thymosin β4 was upregulated specifically in osteosarcoma, colorectal carcinoma, and esophageal cancer. To confirm the over-expression of thymosin β4 in these tumors, we analyzed expression of thymosin β4 with each additional microarray of osteosarcoma, colorectal carcinoma, and esophageal cancer. The significant increased expression of thymosin β4 was observed in osteosarcoma and in colorectal cancer. These results suggest that the expression of thymosin β4 is highly related with tumorigenesis of certain tumors including the osteosarcoma and colorectal cancers.