Recurrence and metastasis are frequently observed after radiotherapy for hepatocellular carcinoma (HCC), although upregulation of matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) induced by radiation has been claimed to be involved, the mechanism is not clarified yet. In the present study, by using MHCC97L, a human HCC cell line with metastatic potential, and its xenograft in nude mice, we found that radiation induced a 48-to 72-h temporary increase in the expression of MMP-2 and VEGF both in vitro and in vivo, but only the in vitro invasiveness of MHCC97L cells was enhanced, while the in vivo metastatic potential of tumors was suppressed. Whereas, 30 days after radiation, when the expression of MMP-2 and VEGF decreased to unirradiated control levels, the in vivo dissemination and metastatic potential of residual tumors have just begun to increase with overexpression of TMPRSS4, which induced loss of E-cadherin through induction of Smad-Interacting Protein 1 (SIP1), an E-cadherin transcriptional repressor, and led to epithelial-mesenchymal transition (EMT). This process was blocked by treatment of siRNA-TMPRSS4. In conclusion, our study revealed novel findings regarding the biphasic effect of radiation on the metastatic potential of residual HCC. Overexpression of TMPRSS4 has a critical role in radiation-induced long-term dissemination and metastasis of residual HCC by facilitating EMT. These findings may provide new clues to suppress the radiation-induced dissemination and metastasis, thereby improve the prognosis of HCC patients.