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Tropomyosin 4 (TPM4) has been found to be dys-regulated, and function as oncogene or anti-oncogene in human cancers. However, there was no report on the clinical significance and biological function of TPM4 in colon cancer. This study was designed to investigate the clinical value and biological function of TPM4 in colon cancer. Thus, we detected the TPM4 expression in colon cancer clinical samples, and conducted the gain-of-function in colon cancer cell lines. In our results, TPM4 mRNA and protein expressions were reduced in colon cancer tissues and cell lines compared with normal colon tissues and colon epithelial cell line, respectively. TPM4 protein low-expression was obviously associated with clinical stage, T classification (invasion depth), N classification (lymph node metastasis), distant metastasis and differentiation. Survival analysis showed low-expression of TPM4 was an unfavorable independent prognostic factor for colon cancer patients. Moreover, the experiments in vitro suggested up-regulated TPM4 expression suppressed colon cancer cell migration, invasion and metastasis-associated gene expression including MMP-2, MMP-9 and MT1-MMP, but had no effect on cell proliferation. In conclusion, TPM4 is associated with clinical progression in colon cancer patient and acts as a tumor suppressor in colon cancer cell.