Patients with advanced hepatocellular carcinoma (HCC) have a poor prognosis, and the development of new therapeutic strategies is necessary. Here we report the efficacy of combination chemotherapy in our biochemical modulation. Synergistic effects of interferon-alpha-2b on 5-fluorouracil or cisplatin were demonstrated in Huh7 cells. The efficacy of methotrexate-5-fluorouracil, cisplatin, and interferon-alpha-2b combination therapy was demonstrated in 34 patients with HCC complicated by major portal vein thrombosis. Among the 29 patients eligible for the study, there were 3 complete responders and 10 partial responders with an overall response rate of 45%. The 2-year survival of the 34 patients was 15%, and the median survival of complete and partial responders was 11 months. There was severe transient hematological toxicity. Eight patients suffered from renal insufficiency, and 4 of them underwent hemodialysis. Although a control study is clearly necessary, our combination therapy induced a good response in the patients with advanced HCC. On the basis of our results, intensive chemotherapy should be attempted in advanced HCC complicated by major portal vein invasion.