Previous studies have demonstrated that tyroserleutide (YSL) inhibits tumor growth in an animal model of hepatocellular carcinoma (HCC). However, its effects on HCC metastasis are still not fully understood. To examine YSL as a novel agent to prevent HCC metastasis, a metastatic human HCC orthotopic nude mouse model of MHCC97L was used. The antitumor and antimetastasis effects of YSL were also evaluated in combination with radiation. Hypoxia and epithelial-mesenchymal transition (EMT)-related molecules were studied. YSL inhibited MHCC97L cell invasion in vitro with or without irradiation. YSL did not significantly inhibit tumor growth but decreased pulmonary metastasis and prolonged life-span for more than 40 days, which correlated with down-regulation of matrix metalloproteinase-2. Radiotherapy inhibited early-stage tumor growth and promoted tumor hypoxia. The re-implanted tumor volume in the radiotherapy group was not significantly different from the control, in which the incidence of lung metastasis increased after radiotherapy (6/6 versus 3/6, P = 0.046); however, YSL inhibited the growth of re-implanted tumor after radiotherapy. Furthermore, YSL at 160 or 320 μg/kg/day almost completely inhibited lung metastasis induced by irradiation (1/6 versus 6/6, P = 0.002 for both dosages). YSL down-regulated hypoxia-inducible factor 1α (HIF-1α) and transmembrane protease serine 4 (TMPRSS4), and inhibited EMT was associated with the antimetastasis capability of YSL. Our data suggest that YSL inhibits the enhanced invasiveness and metastatic potential of HCC induced by irradiation through down-regulation of HIF-1α and TMPRSS4 and inhibition of EMT. YSL may have potential as a new antimetastasis agent for radiotherapy.